Improved methods for reprograming non-pluripotent cells into pluripotent stem cells

ABSTRACT

Provided are chemical inducers of pluripotency (CIP) which include glycogen synthase kinase inhibitors, TGFβ receptor inhibitors, cyclic AMP agonists and S-adenosylhomocysteine hydrolase (SAH) inhibitors or histone acetylators. A method of inducing pluripotency in a partially or completely differentiated cell by using such chemical inducers of pluripotency is also provided. The method includes: (i) contacting a cell with the CIPs for a sufficient period of time to result in reprogramming the cell into a pluripotent stem cell having ESC-like characteristics (CiPSC). Isolated chemically induced pluripotent stem cells (CiPSCs) and their progeny, produced by inducing differentiation of the CiPSCs, can be used in a number of applications, including but not limited to cell therapy and tissue engineering.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 International Application No.PCT/CN2015/095981, filed Nov. 30, 2015, herein incorporated byreferences in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Nov. 19, 2018, as a text file named“HGL_101_ST25.txt,” created on Nov. 6, 2018, and having a size of 16,949bytes is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention generally relates to small molecule compositionsand methods for reprogramming eukaryotic cells into pluripotent cells.

BACKGROUND OF THE INVENTION

Pluripotent stem cells, such as embryonic stem cells (ESCs), canself-renew and differentiate into all somatic cell types. Somatic cellshave been reprogrammed to become pluripotent via nuclear transfer intooocytes or through the ectopic expression of defined factors (Wilmut, etal., Nature, 385:810-813 (1997); Takahashi, et al., Cell, 126:663-676(2006); Yamanaka, et al., Nature, 465:704-712 (2010) and Stadtfeld, etal., Genes Dev., 24:2239-2263 (2010)). However, exogenouspluripotency-associated factors, especially Oct4, are indispensable inthese methods for establishing pluripotency (Zhu, Annu. Rev. Biomed.Eng., 13:73-90 (2011); Li, Cell Res., 21:196-204 (2011) and Li, et al.,Proc. Natl. Acad. Sci. U.S.A., 109:20853-20858 (2012)). Additionally,the requirement for tumorigenic genes like c-Myc in these reprogrammingmethods creates a risk of inducing cancerous cells. Accordingly,reprogramming strategies have raised concerns regarding the clinicalapplications (Saha, et al., Cell Stem Cell, 5:584-595 (2009) and Wu, etal., Cell Biol., 13:497-505 (2011)).

Small molecules which can drive reprogramming of somatic cells intopluripotent cells are disclosed in PCT/CN2014/081961. Small moleculeshave advantages because small molecules more readily penetrate thecells, they are nonimmunogenic, more cost-effective, and more easilysynthesized, preserved, and standardized. There is still a need for amethod of chemically reprogramming non-pluripotent cells intopluripotent cells, that increases the efficiency of reprogramming, forexample, by reducing to total reprogramming time and/or increasing thenumber of reprogrammed cells obtained for the same length of time.

It is an object of the present invention to provide a combination ofsmall molecules which can be used to reprogram partially or completelydifferentiated cells into pluripotent cells.

It is also an object of the present invention to provide a method ofreprogramming partially or completely differentiated cells intopluripotent cells with improved efficiency.

SUMMARY OF THE INVENTION

Compositions and methods are disclosed for improving the efficiency ofchemically inducing non-pluripotent cells into pluripotent cells. Themethods are based on the discovery of an intermediate population ofcells (XEN-like cells) during the reprogramming period, which are primedfor conversion into pluripotency, small molecule combinations thatpreferentially bias partially or completely differentiated cells into aXEN-like state and subsequently reprogram the XEN-like cells intopluripotent cells, and the required replating concentration/density.Thus, by selecting small molecules which bias/enrich the conversion ofpartially or completely differentiated cells into a XEN-like state,small molecules which reprogram the XEN-like cells into pluripotentcells, and the appropriate replating concentration, the efficiency ofreprogramming partially or completely differentiated cells is enhancedin terms of number of colonies obtained and reprogramming time.

Accordingly, small molecule cocktails have been identified which can beused to enhance reprogramming of partially or completely differentiatedcells (including cells that are not genetically engineered to expressone or more markers of pluripotency such as Oct4, and which do notnaturally express Oct4), into a XEN-like state and subsequently, intopluripotent cells. The required chemical inducers of pluripotency (CIPs)include (1) a glycogen synthase kinase (GSK) inhibitor, (2) a TGFβreceptor inhibitor, (3) a cyclic AMP agonist, (4) aS-adenosylhomocysteine hydrolase (SAH) inhibitor, (5) a histoneacetylator/deacetylase inhibitor such as valproic acid (VPA; “V”), (6) aDOT1L methyltransferase inhibitor, (7) a retinoic acid receptor (RAR)agonist, (8) an epigenetic modulator, (9) an inhibitor of histonedemethylation and combinations thereof. The CIPs may be providedseparately or in combination as a CIP composition. One or moreepigenetic modulators and retinoic acid receptor agonists, for example,retinoic receptor ligands may also be administered with the CIPs. Insome preferred embodiments, the CIPs include DZNep as an SAH inhibitor.

In a preferred embodiment, the GSK inhibitor is the aminopyrimidine,CHIR99021 (CHIR; “C”)[6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile];the TGFβ receptor inhibitor is 616452 (“6”)[2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine]; thecAMP agonist is Forskolin (FSK;“F”) and the SAH inhibitor is3-deazaneplanocin A (DZNep; “Z”). Preferred methyltransferase inhibitorsinclude SGC 0946 (“S”)(1-[3-[[[(2R,3S,4R,5R)-5-(4-Amino-5-bromo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl](isopropyl)amino]propyl]-3-[4-(2,2-dimethylethyl)phenyl]urea)and EPZ004777,“1-(3-((((2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)urea(“E”). A preferred RAR agonists include AM 580 (“A”)(4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoicacid). A preferred inhibitor of histone demethylation is tranylcypromine(“T”). A preferred epigenetic modulator is 5-azacytidine (“D”).

Also provided is a method of enhancing reprogramming of a cell of afirst type which is not a pluripotent cell, such as a somatic cell, intoa pluripotent cell. Preferred cells to reprogram include fibroblastcells, adipose-derived stem cells (ADSC), neural derived stem cells andintestinal epithelial cells. In a preferred embodiment the method doesnot include transfecting the cell to be reprogrammed so that itexpresses any of Oct4, KLF4, SOX2, C-Myc or NANOG. In this embodiment,the method also does not include contacting the cell to be reprogrammedwith a polypeptide such as a transcription factors. The method disclosedherein includes the steps of (a) contacting the cell to be reprogrammedwith a first cocktail of CIPs (herein, XEN-Cocktail) for a sufficientperiod of time to bias the cells into a XEN-like cell population; (b)contacting the population of XEN-like cells for a sufficient period oftime to reprogram the cell into a chemically induced pluripotent stemcell (CiPSC) with a second cocktail of CIPS (herein, XEN-CiPSC cocktail)and (c) culturing the cells in 2i-medium. The cells are preferablyreplating during step (a) at a density of about 50,000-100,000 cells perwell in a 6-well plate. The 2i-medium preferably additionally includesN2B27. The method optionally includes selecting for EpCAM (Epithelialcell adhesion molecule)-positive cells after step (a). The reprogrammedcell is identified as a pluripotent cell based on ESC-like propertiessuch as morphology, doubling time, expression of ESC markers such asalkaline phosphatase (AP), nanog, Rex1, Sox2, Dax1, Sall4,undifferentiated embryonic cell transcription factor (Utf1), stagespecific embryonic antigen-4 (SSEA-4), and the ability of the cell todifferentiate into tissues of the three embryonic germ layers. TheCiPSCs are isolated and can be further cultured.

Isolated chemically induced pluripotent stem cells (CiPSCs), are notnaturally occurring pluripotent stem cells. CiPSCs possess ESC-likeproperties such as ESC morphology, doubling time similar to ESC,expression of ESC markers such as alkaline phosphatase (AP), nanog,Rex1, Sox2, Dax1, Sall4, undifferentiated embryonic cell transcriptionfactor (Utf1), stage specific embryonic antigen-4 (SSEA-4), and theability of the cell to differentiate into tissues of the three embryonicgerm layers. CiPSCs are different from ESCs for example, in that theyare not directly derived/isolated from the inner cell mass of ablastocyst. CiPSCs are different from other induced pluripotent sterncells (iPSC) in that they are not engineered to express a transgene suchas genes expressing Oct4, KLF4, SOX2, c-Myc or NANOG, or are notproduced by a process that includes transfecting the cells from whichthey obtained to express any of these transgenes. CiPSCs are alsodifferent from other induced pluripotent stem cells (iPSC) in that theyare not produced by a process that includes contacting non-pluripotentcells with one or more polypeptides such as Klf, Oct, Myc or Sox. In apreferred embodiment, the CiPSCs are not genetically engineered, i.e.,the CiPSCs are not altered by introducing or removing genetic elementsfrom the cells. There is no obvious difference among CiPSCs, iPSCs andESCs. However, CiPSCs disclosed herein can be distinguished from ESC atleast by the methods that are used to generate them i.e., by theirorigin. Where ESC are naturally occurring cells, CiPSCs on the otherhand are not naturally occurring and are obtained by treatingnon-pluripotent cells with a combination of small molecules, asdescribed herein.

The CiPSCs can be cultured or induced to differentiate into cells of adesired type. The CiPSCs and their progeny can be used in a number ofapplications, including but not limited to cell therapy and tissueengineering.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the numbers of iPSC colonies induced from MEFsinfected by SKM (FIG. 1A) or SK (FIG. 1B) plus chemicals or Oct4. Errorbars, mean±SD (n=3 biological repeat wells. FIG. 1C shows RT-PCRanalysis of pluripotency marker genes in MEFs, SKM-FSK-iPSCs and ESCs(R1). FIG. 1D shows genomic PCR analysis of SKM-FSK-iPSCs andSK-FSK-iPSCs. FIGS. 1E and 1F show JRT-PCR (FIG. 1E and genomic PCR(FIG. 1F) analysis of iPSC colonies induced by SKM or SK with chemicalstreatment. Abbreviations: M (2-Me-5HT); D (D4476); V (VPA). Tg indicatesexogenously introduced genes. Scale bars, 100 μM.

FIG. 2 shows mRNA levels of pluripotency-related genes detected byRNA-seq analysis in MEFs, GFP-negative cells, GFP-positive clusters andESCs (R1). GFP-negative cells and GFP-positive clusters were collectedon day 24. Cdh1=E-cadherin.

FIG. 3 is a bar graph showing relative number of iPSC colonies followingtreatment with indicated small molecules. (−) control, DMSO. Error barsindicate s.d (n≥2). Abbreviations: PEG2 (Prostaglandin E12);5-aza-C(5-Azacytidine; NaB (Sodium Butyrate).

FIG. 4A shows numbers of GFP-positive colonies induced after DZNeptreatment on day 36. Error bars, mean±SD (n=2 biological repeat wells).FIG. 4B is a FACS analysis of GFP-positive cells induced from OG-MEFs.Left, the absence of GFP-positive cells in initial MEFs; middle andright, proportion of GFP-positive cells induced by VC6TF (middle) andVC6TFZ (right) on day 44. FIG. 4C is a Schematic diagram illustratingthe process of CiPSC generation. Scale bars, 100 mm. For (FIG. 4A),cells for reprogramming were replated on day 12. FIG. 4D shows mRNAlevels of pluripotency-related genes detected by RNA-seq analysis inMEFs, GFP-positive colonies and ESCs (R1). Unlike mouse ESC colonies,these GFP-positive colonies, which were epithelioid and compact, couldnot maintain in ESC culture condition and were collected on day 36. FIG.4E shows mRNA levels of pluripotency-related genes detected by RNA-seqanalysis in MEFs, CiPSCs and ESCs (R1).

FIGS. 5A-K show optimization of the concentrations and treatmentdurations for individual chemicals in the VC6TFZ condition. FIGS. 5A-Gshow the number of colonies of CiPSC cells as a function of theconcentrations of small molecules as indicated (titrated during CiPSCinduction). FIG. 5H shows Durations of each small molecule. FIGS. 5I-Kshow durations of the chemical combinations. The chemical reprogrammingmedium plus VC6TFZ was replaced with 2i-medium at different time points.Error bars indicate the s.d. (n≥2).

FIGS. 6A-F show validated small molecules improving chemicalreprogramming efficiency or kinetics. FIG. 6A shows validated smallmolecules improving chemical reprogramming efficiency in combinationwith VC6TFZ in MEFs. Chemicals added from day 0 to day 12: PGE2, DY131,RG108, 2-Me-5HT and IBMX; chemicals added after day 12: SF1670, UNC0638and SRT1720. (−) control, DMSO. Error bars indicate the s.d. (n≥2).

FIG. 6B shows the effect of TTNPB on improving chemical reprogrammingefficiency in combination with C6FZ or VC6TFZ in MEFs. CiPSC colonieswere quantified on day 50 and day 40 for chemical reprogramming by C6FZand VC6TFZ, respectively. iPSC colonies were quantified on day 24 andday 16 for OSK- and OSKM-induced reprogramming, respectively. Error barsindicate s.d. (n=3). FIG. 6C shows the effect of TTNPB on improvingchemical reprogramming kinetics in combination with VC6TFZ in MEFs.GFP-positive colonies were quantified on the indicated days. (−)control, VC6TFZ. Error bars indicate s.d. (n=3). FIGS. 6D-F show GenomicPCR analysis for CiPSCs.

FIGS. 7A-D shows genomic PCR and southern blot analysis showing thatCiPSCs were free of transgene contamination. FIGS. 7A and B showsgenomic PCR for two sets of viral vectors used. FIG. 7C is a southernblot analysis to detect viral integration events. DNA probe was designedon psi sequence to target both pLL3.7-ΔU6 and tet-on vectors. CiPSCswere analyzed and Tet-O-iPS, pLL-O-iPS and MEFS were used as controls.FIG. 7D shows the ethidium-bromide stained gel used for the southernblot.

FIGS. 8A-B show the numbers of GFP-positive (FIG. 8A) and CiPSC (FIG.8B) colonies induced by removing individual chemicals from VC6TFZ. Theresults of three independent experiments are shown with different colors(white, gray, and black).

FIG. 9A shows pluripotency marker expression in different clones of CiPScells compared to MEFS, as illustrated by RT-PCR. FIG. 9B shows growthcurves for CiPSCs. FIGS. 9B-D show RT-PCR analysis of pluripotencymarkers in MAF-CiPS cells, ADSC-CiPSC and MNF-CiPSCs.

FIG. 10A shows survival curves of chimeras generated from CiPSCs. n,total numbers of chimeras studied. FIGS. 10B and 10C show pluripotencygenes expression in NSC-CiPSCs (A) and IEC-CiPSCs (B) as measured byquantitative real-time PCR (Error bars, mean±SD, n=3).

FIGS. 11A-B show the expression of pluripotency-related genes (FIG. 11A)and Gata6, Gata4, and Sox17 (FIG. 11B) as measured by real-time PCR.FIGS. 11C-D show expression of Sall4, Sox2, Gata6, Gata4 and Sox17validated by real-time PCR. The fold changes in Sall4, Sox2, Gata6,Gata4 and Sox17 expression on days 4, 8, 12, 16, 20 and 24 (FIG. 11C) orat 12 h (FIG. 11D) compared with the expression in MEFs on day 0. Errorbars indicate the s.d. (n=2). FIGS. 11E-I show the effects of individualand combined chemicals or withdrawing chemicals from VC6TF (or VC6TFZ)on the expression of genes. FIGS. 11E-F shows the effects of individualand combined chemicals on the expression of Sall4 and Sox2 on day 12.FIG. 11G-H show the effects of removing chemicals from VC6TF on theexpression of Sall4 and Sox2 on day 12. FIG. 11I shows the effects ofwithdrawing individual chemicals (CHIR, 616452 and FSK) from VC6TFZ onthe expression of the pluripotency marker genes on day 32. FIG. 11Jshows the effects of withdrawing individual chemicals (CHIR, 616452 andFSK) from VC6TFZ on the expression of Gata6, Gata4 and Sox17 on day 32.Error bars indicate the s.d. (n=2). FIG. 11K shows the expression ofpluripotency-related genes in the presence and absence of DZNep on day32. FIG. 11L shows H3K9 methylation in the presence and absence of DZNepon day 32.

FIGS. 12A-B shows relative expression levels of Sall4 (FIG. 12A) andSox2 (FIG. 12B) in MEFs on day 4 post-transduction validated byreal-time PCR. Error bars indicate the s.d (n=2). FIG. 12C shows Oct4promoter-driven luciferase activity was examined in MEFs transfectedwith Sall4 or/and Sox2 plasmids. Error bars indicate the s.d. (n=3).FIGS. 12D-E show Oct4 activation (FIG. 12D) and numbers of GFP-positiveand iPSC colonies (FIG. 12E) induced by the overexpression of Sall4 andSox2, with C6F removed from VC6TFZ.

FIGS. 13A-D show gene expression changes by the knockdown of Sall4,Gata6, Gata4 or Sox17 on day 24. FIG. 13A shows relative expressionchanges of Sall4, Gata6 and Gata4 by Sall4, Gata6 or Gata4 knockdown.FIG. 13B shows relative expression changes of Sox17, Sall4, Gata6, Gata4and Oct4 by Sox17 knockdown. FIG. 13C shows relative expression changesof Sox17 by the knockdown of Sall4, Gata6 or Gata4. FIG. 13D showsrelative expression changes of Sox2 by the knockdown of Sall4, Gata6 orGata4. Error bars indicate the s.d. (n=2). FIGS. 13E-F show the effectsof Sall4, Gata6 or Gata4 knockdown on the expression of Oct4 and iPSCsformation. FIG. 13E shows Oct4 expression change by Sall4, Gata6 orGata4 knockdown on day 32. Error bars indicate the s.d. (n=2). FIG. 13Fshows numbers of GFP-positive and iPSC colonies when Sall4, Gata6 orGata4 was knockdown during chemical reprogramming. Error bars indicatethe s.d. (n=3). FIG. 13G is a schematic diagram illustrating thestepwise establishment of the pluripotency circuitry during chemicalreprogramming.

FIGS. 14A-B show biological activity of DZNep during chemicalreprogramming. FIG. 14A shows the relative ratios of intracellularlevels SAH to SAM compared to that in MEFs as measured by HPLC analysis.Error bars indicate the s.d. (n=2). FIG. 14B shows the use ofreplacement of DZNep by SAH hydrolase inhibitors (Nep A, Adox and DZA)in combination with VC6TF treatment to induce CiPSC generation. Errorbars indicate the s.d. (n=3). Abbreviations: HPLC (high-performanceliquid chromatography). FIG. 14C shows protein levels of EZH2 wereanalyzed by western blot analysis. FIG. 14D shows numbers of iPSCcolonies induced by VC6TF plus shRNA or DZNep. Error bars indicate thes.d. (n=3). FIG. 14E shows real-time PCR showing that Ezh2 was repressedfollowing shRNA-mediated knockdown of Ezh2. Error bars indicate the s.d.(n=2). FIG. 14F shows the relative intracellular cAMP levels compared tothose in MEFs. FIG. 14G shows the effect of inhibition of adenylatecyclase by 2'S′ddAdo on the number of CiPSC colonies generated byVC6TFZ. (−) control, DMSO. FIG. 14H-J show the effect of replacing FSKwith a cAMP analog (DBcAMP with/without IBMX) accompanied by VC6 T plusDZNep (VC6TZ) treatment, or the phosphodiesterase inhibitors (Rolipramand IBMX) in combination with VC6TZ during CiPSC induction, on chemicalreprogramming. FIG. 14K is a quantification of intracellular cAMP levelsfollowing treatment of MEF, with the indicated chemicals. Error barsindicate the s.d. (n≥2). FIG. 14L shows the effect of 616452concentration during the first 20 days of chemical reprogramming (Errorbars, mean±SD, n=3) in NSC and IEC cells. FIG. 14M shows growth curvesfor CiPSCs. NSC-CiPSC-2 from passage 6; NSC-CiPSC-5 from passage 7;IEC-CiPSC-4 from passage 10; and IEC-CiPSC-10 from passage 7. FIG. 14Nshows CiPSC colonies obtained from different RAR agonists used for onchemical reprogramming of OG MEFs. 13-cis-RA, 2 μM; 9-cis-RA, 2 μM;ATRA, All-trans Retinoic acid, 2 μM; AM 580, 0.01 μM; TTNPB, 2 μM; Ch55, 1 μM.

FIG. 15A is a schematic representation of the major steps in thedevelopment of chemical reprogramming systems. The blue hexagonsrepresent reprogramming transcription factors, and the red squaresrepresent the major small molecules identified at each step.Reprogramming boosters are displayed on the right, corresponding todifferent reprogramming conditions. The alternative Oct4 substitutesD4476 and 2-Me-5HT are displayed below FSK. Abbreviations: O (Oct4), S(Sox2), K (Klf4), M (c-Myc), Trany1 (Tranylcypromine). FIGS. 15B and 15Cshow expression of Sall4, Gata4 and Sox17 genes at the early stage ofchemical reprogramming from NSCs (FIG. 15B) and IECs (FIG. 15C) at day 0(D0)), day 4 (D4) and day 16 (D16), respectively, measured byquantitative real-time PCR. FIGS. 15D and 15F show expression of Sall4,Gata4 and Sox17 genes by the chemical cocktail with differentconcentration of 616452 at day 16 (FIG. 15F) and day 20 (FIG. 15D)(error bars, mean±SD, n=3). FIG. 15E shows expression of pluripotencygenes Sall4, Lin28, Esrrb, Dppa2 and Oct4 during the chemicalreprogramming from NSC (day 0 (D0), day 4 (D4), day 12 (D12), day 16(D16) and day 20 (D20), respectively) measured by quantitative real-timePCR. FIG. 15G shows mRNA levels of Sall4, Gata4, Gata6 and Sox17 genesdetected by RNA-seq analysis in MEFs, IECs and NSCs.

FIG. 16A shows numbers of CiPSC colonies generated from the inside(blue) and outside (red) of epithelial colonies in 8 batches ofexperiments. For experiments #5 and #7, the cell confluence ofepithelial colonies was less than 20%. FIG. 16B shows qRT-PCR analysisof XEN cell markers (Gata4, Gata6, Sox17, Sox7, Sall4) and pluripotencymarker Oct4 in MEFs, cells at the end of stage 1 (day 16) and stage 2(day28) and eXEN (embryo-derived XEN cells). FIG. 16C shows CiPSC colonynumbers generated at the end of stage 3 from EpCAM-negative (−),EpCAM-positive (+) and total cell populations sorted at day 20 (in stage2 of chemical reprogramming). FIG. 16D shows XEN-like colony numbers atday 16 generated from EpCAM positive (+), negative (−) populations andtotal cells after FACS sorting at day 12 (in stage 1 of chemicalreprogramming). Error bars indicate the SD (n≥2).

FIG. 16E shows CiPSC colony numbers induced from neural stem cells (NSC,left) from indicated populations and intestinal epithelium cells (IEC,right). EpCAM negative (−), positive (+) and total populations weresorted by FACS at day 13 (for IECs) and day 20 (for NSCs), respectively.

FIG. 17A shows numbers of SALL4 and GATA4 double-positive colonies aftertreatment with DMSO and VC6TF (CHIR, 10 μM and 20 μM) for 12 days. FIG.17B shows qRT-PCR analysis of XEN cell markers (Gata4, Gata6, Sox17,Sall4) expression after treatment with VC6TF (CHIR, 10 μM and 20 μM) for12 days. eXEN was set as a positive control. FIG. 17C shows qRT-PCRanalysis of MET markers (EpCAM, Cdh1) expression after treatment ofVC6TF (CHIR, 10 μM and 20 μM) for 12 days. FIG. 17D shows numbers andphase images of XEN-like colonies after treatment with control cocktail(VC6TF with CHIR, 20 μM) and that with additional small moleculeEPZ004777 (E) and AM580 (A) for 16 days. Cells were re-plated at day 12by 1:2. FIG. 17E shows qRT-PCR analysis of Sall4 expression in cellstreated with different small-molecule cocktails during stage 1. FIG. 17Fshows qRT-PCR analysis of XEN cell markers (Gata4, Gata6, Sox17, Sox7and Sall4) expression induced with VC6TF (CHIR, 20 μM) and AM580 (A),EPZ004777 (E) or A plus E for 12 days. eXEN was set as a positivecontrol. FIG. 17G shows numbers of SALL4 and GATA4 double-positivecolonies after treatment with VC6TF (CHIR, 20 μM), and that with AM580,EPZ004777 (EPZ) and with their combination for 12 days. FIG. 17H showsqRT-PCR analysis of MET markers (Cdh1, EpCAM) expression after treatmentwith VC6TF (CHIR, 20 μM) and that with AM580 (A), EPZ004777 (E) and Aplus E for 12 days. FIG. 17I shows qRT-PCR analysis of Gata4, Gata6,Sox17, Lin28a, EpCAM and Cdh1 expression in cells treated by differentsmall molecule cocktails during stage 1. eXEN and CiPSCs were set ascontrols. Error bars indicate the SD (n≥2).

FIG. 18A shows numbers of CiPSC colonies at day 44 induced with acontrol cocktail (VC6TFZ) and with 5-aza-dC, 5-aza-dC plus EPZ004777(EPZ) or SGC0946 (SGC) in stage 2 of 12 days. FIGS. 18B and C shownumber of CiPSC colonies with treatment of control (VC6TFZ) and withEPZ004777 (EPZ) or SGC0946 (SGC) in stage 2 and numbers of CiPSCcolonies in 2i-medium with different components in stage 3. FIG. 18Dshows Numbers of CiPSC colonies at the end of reprogramming fromdifferent densities of cells in each well (6-well plate) re-plated atday 12. #1 and #2 were two independent experiments. FIG. 18E showsNumbers of CiPSC colonies at the end of reprogramming, with a differenttime course in stage 2. Error bars indicate the SD (n≥3). FIG. 18F showsthe effect of the concentration (left) and duration (right) of 5-aza-dC.5-aza-dC added in stage 2. FIG. 18G shows the effect the concentration(left) and duration (right) of SGC0946 (SGC). SGC was added in stage 2.FIG. 18H shows percentage of XEN-like colonies that reprogrammed toCiPSC colonies at day 40 by new protocol in 5 batches of experiments.FIG. 18I shows numbers of CiPSC colonies generated inside (blue) andoutside (red) XEN-like colonies by new protocol in 3 batches ofexperiments. Error bars indicate the SD (n≥2).

FIG. 19A is a schematic comparison of the new protocol in this study andthe protocol disclosed in Hou et al., 2013. In total, 1-40 CiPSCcolonies (in 2 wells) were induced from initial 50,000 fibroblasts in a60-day induction using the initial protocol. Whereas 1,000-9,000 CiPSCcolonies (in 10-15 wells) were obtained from initial 50,000 fibroblastsafter 40 days of small-molecule treatment by using the new protocol.FIG. 19B is a comparison of the previously described protocol (noXEN-like state bias) and the protocol which takes advantage of theCEN-like state, in primary CiPSC colony numbers (day 40) induced frommouse neonatal fibroblasts (MNFs, left) and mouse adult fibroblasts(MAFs, right), respectively. FIG. 19C shows Numbers of CiPSC coloniesgenerated under shortened durations for each stage. For example,“12+10+6” represents a sequential duration of 12 days for stage 1, 10days for stage 2, and 6 days for stage 3. The minimal time courserequired for CiPSC induction was 26 days (12+8+6), by which one CiPSCcolony was obtained. FIG. 19D shows Pluripotency marker expression inCiPSC colonies induced by the new protocol, analyzed by qRT-PCR.

FIG. 20A shows Dynamic expression change in pluripotency-associatedgenes (Oct4, Sox2, Nanog, Sall4, Lin28a, Zfp42, Dppa2, Esrrb) and METrelated genes (Cdh1, EpCAM) at the indicated time points during chemicalreprogramming, examined by qRT-PCR. FIG. 20B is a scatter plot diagramof single cell qRT-PCR analysis in the expression of Sall4 and Gata4,Gata4 and Sox17, and Sall4 and Sox2, respectively, at the indicated timepoints. Gene expression levels were indicated by log 2 (fold change tothe minimal level in these samples). FIG. 20C is a Comparison ofXEN-related gene expression pattern at indicated time points duringchemical reprogramming (upper) and OSKM-induced reprogramming (bottom)measured by qRT-PCR. CiPSCs, 4F-iPSCs, ESCs and eXEN were set ascontrols. FIG. 20D is qRT-PCR analysis of some pluripotency-associatedgenes in the cells treated with different cocktails as indicated instage 2. FIG. 20E is a schematic representation of the two routes ofsomatic reprogramming by using chemical approach (left) and transgenicapproach (right). Error bars indicate biological repeats and the SD(n≥2).

FIG. 21A shows numbers of SALL4 and GATA4 double-positive colonies withSall4, Gata4, Gata6 or Sox17 knockdown at day 12 of chemicalreprogramming. Non-targeting vector shRNA (shControl) was used asnegative control. Sh1 and sh2 represent two shRNA vectors for each gene.FIG. 21B shows the expression of Oct4 on day 28 with Sall4, Gata4, Gata6or Sox17 knockdown measured by qRT-PCR relative to that treated with anon-targeting vector (shControl). FIG. 21C shows numbers of CiPSCcolonies with Sall4, Gata4, Gata6 or Sox17 knockdown. FIG. 21D shows.The expression of Sall4, Gata4, Gata6 and Sox17 with Sall4, Gata6, Gata4or Sox17 knockdown measured by qRT-PCR on day 16, relative to that inthe non-targeting vector (shControl). FIG. 21E shows the expression ofSox2 with Sall4, Gata6, Gata4 or Sox17 knockdown measured by qRT-PCR onday 28, relative to that in the non-targeting vector (shControl). FIG.21F shows numbers of iPSC colonies induced from neural stem cells (NSCs)and intestinal epithelium cells (IECs) with Sall4, Gata4, Gata6 or Sox17knockdown. FIG. 21G shows numbers of iPSC colonies induced by OSKM withSall4, Gata4, Gata6 or Sox17 knockdown. FIG. 21H shows numbers ofXEN-like colonies by overexpression of SALL4 (S4), GATA4 (G4), GATA6(G6) or their combinations in the presence of small-molecule cocktailVTAE (withdrawal of C6F from VC6TFAE) treatment. (−) represents cellstreated with VTAE without the overexpression of XEN related genes.DMSO-treated cells are shown as negative controls. FIG. 21I showsqRT-PCR analysis of XEN cell markers Sall4, Gata4, Sox17, Gata6 and Sox7by treatment of VC6TFAE or overexpression of Sall4 (S4) plus Gata4 (G4)or Gata6 (G6) in the presence of VTAE. eXEN was set as a positivecontrol. FIG. 21J shows qRT-PCR analysis of Oct4 induced byoverexpression of SALL4, GATA4, GATA6 and their combination in thepresence of VTAE on day 20. FIG. 21K shows the expression of Sox2 by theoverexpression of SALL4 (S4), GATA4 (G4), GATA6 (G6) and SALL4 plusGATA6 or GATA4 in the presence of VTAE. FIG. 21L shows numbers of iPSCcolonies generated by Dox-induced expression of Sox2 in the indicatedtime courses, with the expression of Sall4 plus Gata4 or Gata6 in thepresence of VTAEZ. Error Bars Indicate the SD (n≥2).

FIG. 22A shows relative expression of XEN-related genes in differentcell types as indicated measured by qRT-PCR. eXEN-1 and eXEN-2 were twosublines of eXENs, maintained in traditional XEN culture medium (Kunathet al., 2005) and stage 1 medium of chemical reprogramming,respectively. FIG. 22B shows hierarchical clustering of global geneexpression profiles in different cell types. XEN-like cell samples atdifferent time points (day 16, 20, 26 and 28) during chemicalreprogramming were indicated as D16, D20, D26 and D28. Controls were twobatches of MEFs (MEFs-1, MEFs-2), eXEN-1, eXEN-2, two CeXEN cell lines(CeXEN-1, CeXEN-2), two CiPS cell lines (CiPSCs-1, CiPSCs-2) and two EScell lines (ESCs-1, ESCs-2). FIG. 22C shows qRT-PCR analysis of EpCAM,Cdh1 and Sox2 in different types of XEN cells as indicated. MEFs andESCs were set as controls. FIG. 22D shows qRT-PCR analysis ofpluripotency marker expression in two CiPSC colonies induced from eXENand two CiPSC colonies induced from CeXEN. MEFs and ESCs were set ascontrols. Error bars indicate the SD (n≥2).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “chemically induced pluripotent stem cells” (CiPSCs) as usedherein refers to pluripotent cells derived from a cell that is notpluripotent, i.e., a multipotent or differentiated cells, by contactingthe non-pluripotent cell with chemical compounds, not by expression ofone or more transfected genes.

As used herein a “culture” means a population of cells grown in a mediumand optionally passaged. A cell culture may be a primary culture (e.g.,a culture that has not been passaged) or may be a secondary orsubsequent culture (e.g., a population of cells which have beensubcultured or passaged one or more times).

As used herein “enhancing”, or “increasing” the efficiency ofreprogramming means reducing to total reprogramming time and/orincreasing the number of reprogrammed cells obtained from the samestarting cell density the same length of time when compared to achemical reprogramming method that does not proceed via biasing thecells to be programming towards a XEN-like state.

The term “Induced pluripotent stem cell” (iPSC), as used herein, is atype of pluripotent stem cell artificially derived from anon-pluripotent cell. CiPSCs are iPSCs; however, they differ from someiPSCs in that they are not genetically engineered.

The term “isolated” or “purified” when referring to CiPSCs meanschemically induced pluripotent stem cells at least 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% free ofcontaminating cell types such as non-pluripotent cells. The isolatedstem cells may also be substantially free of soluble, naturallyoccurring molecules.

The term “pluripotency” (or pluripotent), as used herein refers to astem cell that has the potential to differentiate into any of the threegerm layers: endoderm (for example, interior stomach lining,gastrointestinal tract, the lungs), mesoderm (for example, muscle, bone,blood, urogenital), or ectoderm (for example, epidermal tissues andnervous system). The term “not pluripotent” means that the cell does nothave the potential to differentiate into all of the three germ layers. Amultipotent stem cell is less plastic and more differentiated, and canbecome one of several types of cells within a given organ. For example,multipotent blood stem cells can develop into red blood cellprogenitors, white blood cells or platelet producing cells. Adult stemcells are multipotent stem cells. Adipose-derived stem cells aremultipotent.

“Reprogramming” as used herein refers to the conversion of a onespecific cell type to another. For example, a cell that is notpluripotent can be reprogrammed into a pluripotent cell. Where thenon-pluripotent cell is reprogrammed into a pluripotent cell usingchemical compounds, the resulting cell is a chemically inducedpluripotent stem cell.

“Reprogramming medium” as used herein refers to cell culture medium thatincludes one or more chemical inducers of pluripotency.

“2i medium” as use herein refers to ESC culture medium with dualinhibition of glycogen synthase kinase-3 and mitogen-activated proteinkinase signaling, for example, ESC culture medium supplemented with 2i(CHIR99021 and PD0325901).

The term “small molecule” refers to a molecule, such as an organic ororganometallic compound, with a molecular weight of less than 2,000Daltons, more preferably less than 1,500 Daltons, most preferably lessthan 1,000 Daltons.

“XEN-like cells” are used herein refers to cells which are characterizedas epithelial cells, and which express XEN markers such as SALL4, GATA4and SOX17. XEN-like state when used connection with cells refers toexpression of one or more XEN markers.

II. Compositions

A. Small Molecules Inducing Pluripotency

Chemical compounds that induce pluripotency i.e., chemical inducers ofpluripotency (CIP) include small molecules having a molecular weight ofless than 2,000 Daltons, more preferably less than 1,500 Daltons, mostpreferably less than 1,000 Dalton, alone or in combination withproteins. The small molecules may have a molecular weight less than orequal to 900 Daltons or, less than or equal to 500 Daltons. Largermolecules can be used in chemically-induced reprogramming, preferablytargeting the same pathway as the small molecules identified here.Several protein factors, such as recombinant bFGF, have beendemonstrated to be effective in the following protocol for chemicalreprogramming.

Accordingly, small molecule cocktails have been identified which can beused to enhance reprogramming of partially or completely differentiatedcells (including cells that are not genetically engineered to expressone or more markers of pluripotency such as Oct4, and which do notnaturally express Oct4), into a XEN-like state and subsequently, intopluripotent cells. The required chemical inducers of pluripotency (CIPs)include (1) a glycogen synthase kinase (GSK) inhibitor, (2) a TGFβreceptor inhibitor, (3) a cyclic AMP agonist, (4) aS-adenosylhomocysteine hydrolase (SAH) inhibitor, (5) a histoneacetylator such as valproic acid (“V”), (6) a DOT1L methyltransferaseinhibitor, (7) a retinoic acid receptor (RAR) agonist, (8) an epigeneticmodulator, (9) an inhibitor of histone demethylation and combinationsthereof. The CIPs may be provided separately or in combination as a CIPcomposition. One or more epigenetic modulators and retinoic acidreceptor agonists, for example, retinoic receptor ligands may also beadministered with the CIPs. In some preferred embodiments, the CIPsinclude DZNep as an SAH inhibitor.

(1). GSK Inhibitors

The preferred GSK inhibitor is the aminopyrimidine, CHIR99021 having thechemical name[6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile](“C”), used in a concentration of about 20 μM. Other GSK inhibitors canalso be used in the methods disclosed herein, and they include, but arenot limited to BIO-acetoxime (for example 1 μM); GSK 31 inhibitor XV;SB-216763; CHIR 99021 trihydrochloride, which is the hydrochloride saltof CHIR99021; GSK-3 Inhibitor IX [((2Z,3E)-6′-bromo-3-(hydroxyimino)-[2,3′-biindolinylidene]-2′-one]; GSK 3 IX[6-Bromoindirubin-3′-oxime]; GSK-3β Inhibitor XII[3-[[6-(3-Aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol];GSK-3 Inhibitor XVI[6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-pyrimidin-2-ylamino)ethyl-amino)-nicotinonitrile];SB-415286 [3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione]; and Bio [(27,3′E)-6-bromoindirubin-3′-oxime], usedat a concentration equivalent to 20 μM CHIR99021.

(2). TGFβ Receptor Inhibitor

The TGFβ inhibitor is preferably inhibits the TGFβ type 1 receptoractivating receptor-like kinase (ALK) 5 in some embodiments, and canadditionally inhibit ALK 4 and the nodal type receptor 1 receptor ALK7in other embodiments

The preferred TGFβ receptor inhibitor is 616452. Other TGFβ inhibitorsare known in the art and are commercially available. Examples includeE-616452[2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine]; A83-01[3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide];SB 505124[2-[4-(1,3-Benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine];GW 788388[4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide];and SB 525334[6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline],and dorsomorphine.

(3) cAMP Agonists

The preferred cAMP agonist is Forskolin (F). However, any cAMP agonistcan be included in the cocktail of CINPs disclosed herein. Examplesinclude, but are not limited to prostaglandin E2 (PGE2), rolipram,genistein and cAMP analogs such as DBcAMP or 8-bromo-cAMP.

(4). SAH Inhibitors

The preferred SAH inhibitor is 3-deazaneplanocin A (DZNep; “Z”). Otheruseful SAH hydrolase inhibitors that can be included in the CIPcombination compositions disclosed herein include, but are not limitedto, (−) Neplanocin A (NepA), Adenozine periodate (oxidized) Adox and3-deazaadenosine (DZA) and combinations thereof

(5). Histone Acetylator/Deacetylase Inhibitors

The preferred histone acetylator is valproic acid. However, otherhistone deacetylase inhibitors are commercially available and can beused. Non-limiting examples include apicidin, CI 994 (N-acetyldinaline4-(Acetylamino)-N-(2-aminophenyl)benzamide), Depsipeptide, KD 5170(S-[2-[6-[[[4-[3-(Dimethylamino)propoxy]phenyl]sulfonyl]amino]-3-pyridinyl]-2-oxoethyl]ethanethiocacid ester), sodium, 4-pehynl butyrate, sodium butyrate, UF 010, etc.

(6). DOT1L Methyltransferase Inhibitors

DOT1L methyltransferase inhibitors are preferred. Preferred examplesinclude methyltransferase inhibitors include SGC 0946 (“S”) andEPZ004777 (“E”).

(7). Retinoic Acid Receptor (RAR) Agonists

Ch 55([4-[(1E)-3-[3,5-bis(1,1-Dimethylethyl)phenyl]-3-oxo-1-propenyl]benzoicacid], a highly potent synthetic retinoid that has high affinity forRAR-α and RAR-β receptors and low affinity for cellular retinoic acidbinding protein (CRABP)]; AM580([4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoicacid]; an analog of retinoic acid that acts as a selective RARαagonist);[4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid] (TTNPB).

Preferred RAR agonists include AM 580 (“A”) and Ch 55.

(8). Epigenetic Modulators

Epigenetic modulators that can be included in the CIP compositioninclude one or more of 5-azacytidine, decitabine and RG108 andcombinations thereof. A preferred epigenetic modulator is 5-azacytidine(“D”).

(9). Inhibitors of Histone Demethylation

A preferred inhibitor of histone demethylation is tranylcypromine (“T”).Tranylcypromine is a nonselective and irreversible monoamine oxidaseinhibitor (MAOI). Another useful MAOI which are also inhibitors ofhistone demethylation include phenelzine (Lee, et al. Chem and Biol.,13:563-567 (2006), Additional non-limiting examples include compoundXZ09 disclosed in Zhou, et al., Chem Biol. and Drug Design,85(6):659-671 (2015) and nonpeptide propargylamines (Schmidttt, et al.J. Med. Chem., 56 (18), pp 7334-7342 (2013).

(10). Additional Small Molecule Boosters

In some embodiments, small molecules that facilitate late reprogrammingand small molecules that improve/boost chemical reprogramming efficiencyover the levels seen with VC6TFZ are included. Improved/boostedefficiency can be manifested by reducing the time needed to generatesuch pluripotent cells (e.g., by shortening the time to development ofpluripotent cells by at least a day compared to a similar or sameprocess without the small molecule). Alternatively, or in combination, asmall molecule can increase the number of pluripotent cells generated bya particular process (e.g., increasing the number in a given time periodby at least 10%, 50%, 100%, 200%, 500%, etc. compared to a similar orsame process without the small molecule).

Small molecules that improve/boost chemical reprogramming efficiencyinclude [N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide](SF1670);[N-(4-(Diethylaminobenzylidenyl)-N-(4-hydroxybenzoyl)-hydrazine](DY131);[2-Cyclohexyl-6-methoxy-N-[1-(1-methylethyl)-4-piperidinyl]-7-[3-(1-pyrrolidinyl)propoxy]-4-quinazolinamine](UNC0638);[N-(2-(3-(piperazin-1-ylmethyl)imidazo[2,1-b]thiazol-6-yl)phenyl)quinoxaline-2-carboxamidehydrochloride] (SRT1720); 2-Me-5HT (“2M5” 2-methyl-5-hydroxytryptamine);and [3,7-Dihydro-1-methyl-3-(2-methylpropyl)-1H-purine-2,6-dioneand](IBMX) and D4476 (D4476 (CAS 301836-43-1)(4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide),a high purity Casein kinase inhibitor and TGF-β type-I receptor (ALK5)inhibitor). An example of small molecule combinations used to reprogramcells is shown in Table 1A. Table 1A small molecule combination forinducing pluripotency

small molecule combination that can Initial cell CiPSC inducepluripotency numbers colony CHIR99021 + 616452 + Forskolin 100,000 1CHIR99021 + 616452 + Forskolin + 50,000 3 DZNep CHIR99021 + 616452 +Forskolin + 40,000 8 DZNep + VPA CHIR99021 + 616452 + Forskolin + 28,00042 DZNep + TTNPB CHIR99021 + 616452 + Forskolin + 90,000 1 DZNep +Tranylcypromine CHIR99021 + 616452 + Forskolin + 50,000 1 VPA +Tranylcypromine CHIR99021 + 616452 + DZNep + 300,000 1 VPA +Tranylcypromine CHIR99021 + 616452 + Forskolin + 20,000 2 DZNep + 4PB +Tranylcypromine CHIR99021 + 616452 + Forskolin + 40,000 12 DZNep + VPA +Tranylcypromine (VC6TFZ) CHIR99021 + 616452 + DBcAMP + 40,000 1 DZNep +VPA + Tranylcypromine CHIR99021 + 616452 + IBMX + 40,000 1 DZNep + VPA +Tranylcypromine CHIR99021 + 616452 + Rolipram + 40,000 1 DZNep + VPA +Tranylcypromine CHIR99021 + 616452 + Forskolin + 50,000 32 NepA + VPA +Tranylcypromine CHIR99021 + 616452 + Forskolin + 50,000 28 Adox + VPA +Tranylcypromine CHIR99021 + 616452 + Forskolin + 50,000 10 DZA + VPA +Tranylcypromine CHIR99021 + 616452 + Forskolin + 30,000 14 Decitabine +EPZ + VPA + Tranylcy- promine CHIR99021 + 616452 + Forskolin + 40,000 20DZNep + VPA + Tranylcypromine + TTNPB CHIR99021 + 616452 + Forskolin +40,000 25 DZNep + VPA + Tranylcypromine + AM580 CHIR99021 + 616452 +Forskolin + 40,000 19 DZNep + VPA + Tranylcypromine + Ch55 TD114-2 +616452 + Forskolin + 40,000 40 DZNep + VPA + Tranylcypromine CHIR99021 +616452 + Forskolin + 300,000 6 VPA + Tranylcypromine + 2M5 + D4476 +Butyrate + UNC0638 + Scriptaid CHIR99021 + 616452 + Forskolin + 250,0005 VPA + Tranylcypromine + TTNPB + PGE2 + 5-aza-C CHIR99021 + 616452 +Forskolin + 250,000 8 VPA + Tranylcypromine + TTNPB + PGE2 + DecitabineCHIR99021 + 616452 + Forskolin + 30.000 14 VPA + Tranylcypromine +Decitabine + EPZ

Concentration ranges for exemplary small molecules that can be includedin the formulations disclosed herein are provided in Table 1B.

Table 1B Summary of Small Molecule Concentrations

preferred Chemical Concentration concentrations names ranges μMCHIR99021 0.1-40  20 616452 0.1-50  10 *Forskolin  0.1-100 10 or 50DZNep 0.005-0.5  0.05 VPA  50-2000 500 SGC 0946  2-10 5 TTNPB 0.01-5   2Tranylcypromine  1-40 5 4PB 0.1-50  2 DBcAMP  0.1-500 50 IBMX  0.1-50050 Rolipram 0.1-50  10 NepA 0.01-5   0.05 Adox 0.1-50  10 DZA 0.1-50  10Decitabine 0.01-5   0.1 EPZ 0.1-20  5 AM580 0.01-5   0.05 Ch55 0.01-5  2 TD114-2 0.1-20  2 2M5 0.1-40  5 D4476 0.1-40  5 Butyrate  1-400 200UNC0638 0.01-5   0.5 Scriptaid 0.01-5   0.5 PGE2 0.1-20  5 5-aza-C0.01-50   5 RG108 0.01-100  10 SRT1720 0.1-20  2 *50 μM Forskolin ispreferred in a XEN-cocktail: 10 μM forskolin if preferred in a XEN-XiPSCcocktail.

B. Protein Factors

Protein factors, such as recombinant basic fibroblast growth factor(bFGF), have been demonstrated to be effective in the following protocolfor chemical reprogramming. bFGF can be used in a concentration rangefrom 10 ng/mL-200 ng/mL, preferably at concentration of 100 ng/mL.

C. Cells to be Induced

The induced pluripotent stem cells are obtained by inducing partially orcompletely differentiated cells obtained from a mammal such as anymammal (e.g., bovine, ovine, porcine, canine, feline, equine, primate),preferably a human. Sources include bone marrow, fibroblasts, fetaltissue (e.g., fetal liver tissue), peripheral blood, umbilical cordblood, pancreas, skin or any organ or tissue. In a preferred embodiment,the CiPSCs are obtained from chemically induced fibroblasts,adipose-derived stem cells, neural stem cells or cells from theintestinal epithelium. In a more preferred embodiment, CiPSCs areobtained from chemically induced neonatal (for example foreskin) oradult fibroblasts. However, CiPSCs can be obtained from other cell typesincluding but not limited to: multipotent stem cells, cells ofhematological origin, cells of embryonic origin, skin derived cells,fibroblasts, adipose cells, epithelial cells, endothelial cells,mesenchymal cells, parenchymal cells, neurological cells, and connectivetissue cells. multipotent stem cells, cells of hematological origin,cells of embryonic origin, skin derived cells, fibroblasts, adiposecells, epithelial cells, endothelial cells, mesenchymal cells,parenchymal cells, neurological cells, and connective tissue cells. Thecell to be reprogrammed can be obtained from a sample obtained from amammalian subject. The subject can be any mammal (e.g., bovine, ovine,porcine, canine, feline, equine, primate), including a human. The sampleof cells may be obtained from any of a number of different sourcesincluding, for example, bone marrow, fetal tissue (e.g., fetal livertissue), peripheral blood, umbilical cord blood, pancreas, skin or anyorgan or tissue.

In a preferred embodiment, the CiPSCs are obtained from fibroblasts andadipose-derived stem cells. In a more preferred embodiment, CiPSCs areobtained from fibroblast, which can be neonatal (for example foreskinfibroblasts) or adult fibroblast. In still another preferred embodiment,the non-pluripotent cells do not express Oct4 and/or are not geneticallyengineered to express one or more markers of pluripotency.

Cells may be isolated by disaggregating an appropriate organ or tissuewhich is to serve as the cell source using techniques known to thoseskilled in the art. For example, the tissue or organ can bedisaggregated mechanically and/or treated with digestive enzymes and/orchelating agents that weaken the connections between neighboring cells,so that the tissue can be dispersed to form a suspension of individualcells without appreciable cell breakage. Enzymatic dissociation can beaccomplished by mincing the tissue and treating the minced tissue withone or more enzymes such as trypsin, chymotrypsin, collagenase,elastase, and/or hyaluronidase, DNase, pronase, dispase etc. Mechanicaldisruption can also be accomplished by a number of methods including,but not limited to, the use of grinders, blenders, sieves, homogenizers,pressure cells, or insonators.

D. Chemically Induced Pluripotent Stem Cells (CiPSCs)

CiPSCs are physiologically and morphologically indistinguishable fromEmbryonic Stem Cells (ESC). The Examples show that CiPSCs grow with adoubling time similar to ESC, and like ESC, express pluripotencymarkers, have a similar gene expression profile to ESC, and have asimilar DNA methylation and histone modifications at Oct4 and Nanogpromoters. Karyotyping analysis also demonstrates that CiPSCs do notacquire chromosomal abnormalities. Further evidence that the CiPSCs arepluripotent is their ability to differentiate into tissues of the threeembryonic germ layers. These findings demonstrate the ability tomanipulate differentiated human cells to generate an unlimited supply ofpatient-specific pluripotent stem cells.

CiPSCs possess ESC-like properties such as ESC morphology, doubling timesimilar to ESC, expression of ESC markers such as alkaline phosphatase(AP), nanog, Rex1, Sox2, Dax1, Sall4, undifferentiated embryonic celltranscription factor (Utf1), stage specific embryonic antigen-4(SSEA-4), and the ability of the cell to differentiate into tissues ofthe three embryonic germ layers. Such cells can also be characterized bythe down-regulation of markers characteristic of the differentiated cellfrom which the CiPSC is induced. For example, CiPSCs derived fromfibroblasts may be characterized by down-regulation of the fibroblastcell marker Thy1 and/or up-regulation of SSEA-1. There is no minimumnumber of pluripotency markers that must be displayed on CiPSCs. Thegold standard for pluripotency is the differentiation potential intocell types of all three germ layers. Teratoma assay, chimeras assay andthe germ-line transmission capability are some direct assays to testtheir differentiation potential.

III. Methods of Making

A. Induction of CiPSCs

CiPSCs can be induced by providing partially or completelydifferentiated cells in a culture media containing the CIPs for asufficient period of time to result in reprogramming the cells intochemically induced pluripotent stem cell (CiPSC). The reprogrammed cellsare defined as pluripotent cells based on possession of ESC-likeproperties such as morphology, doubling time, expression of ESC markersfor example alkaline phosphatase (AP); nanog, Rex1; Sox2; Dax1; Sall4;undifferentiated embryonic cell transcription factor (Utf1); stagespecific embryonic antigen-4 (SSEA-4), and the ability of the cell todifferentiate into tissues of the three embryonic germ layers.

The CIP compounds are contacted with the cells to be induced in anamount effective to induce and/or enhance reprogramming ofnon-pluripotent cells into pluripotent cells. One of skill in the artcan readily determine the concentrations of the CIP compounds disclosedherein required to provide complete reprogramming using methods outlinedin the examples below, or other methods known in the art. In somepreferred embodiments, the CIPs include an SAH inhibitor.

VPA is administered to the cells to a concentration between 500 μM and0.5 mM, CHIR is administered to a concentration between 10 to 20 μMpreferably, 20 μM, 616452 is administered to a concentration between 5to 10 μM, FSK is administered to a concentration between 10 to 50 μM andDZNep is administered to a concentration between 20 nM and 0.1 μM,preferably, between 0.05 to 0.1 μM, and more preferably, between 20 and200 nM. Exemplary combinations of small molecules that can be used toinduce pluripotency in a non-pluripotent cell and concentration rangesare provided in Tables 1A and B.

616452 and Forskolin need to be present the entire time before the useof 2i-medium. CHIR99021 should be used in the first 12 days, and ispreferably present the entire time before the use of 2i-medium. DZNepshould be added at the late stage of reprogramming (day 12 to day 40),preferably, day 16 after the initial treatment of other small molecules.The small molecule combination should be changed into 2i-medium after atime point between day 26 and day 48, preferably day 28. Different celltypes have different optimal concentrations of small molecules. Thesecan be determined by routine experimentation based on the studiesdescribed herein. The order of exposure and the period of time ofexposure are similar between cell types.

In a preferred embodiment, the method includes culturing cells in areprogramming medium containing the CIPs, and further culturing thecells in an ESC culture medium for more than 4 days with dual inhibitionof glycogen synthase kinase-3 (GSK3) and mitogen-activated proteinkinase (MAPK) signaling after about day 28 post treatment with thereprogramming medium. In one embodiment, dual inhibition of GSK3 andMAPK is accomplished using CHIR99021 and PD0325901.

In some embodiments, the method further includes contacting the cellswith additional small molecules that facilitate late reprogramming forexample, cAMP agonists other than forskolin and/or epigenetic modulatorsdisclosed herein, and/or small molecules that improve/boost chemicalreprogramming efficiency disclosed herein. Epigenetic modulators can beincluded in the composition containing VC6TF. Alternatively, these smallmolecules can be included in cell culture medium following treatment ofthe cells with VC6TF. The cells are preferably exposed to the smallmolecules for more than 1 day. In some embodiments, treatment of cellswith cAMP agonists and epigenetic modulators does not exceed the periodof treatment with VC6TF. In other embodiments treatment of cells withcAMP agonists and epigenetic modulators does not exceed the period oftreatment with VC6TFZ. In still other embodiments, treatment of cellswith cAMP agonists and epigenetic modulators does not exceed the periodof treatment with VC6TF plus VC6TFZ. A preferred small molecule forboosting chemical reprogramming efficiency is TTNPB.

The disclosed methods yield induced pluripotent stem cells without theneed to transfect cells with genes such as Oct4, KLF4, SOX2, C-Myc orNANOG or the need to contact the cells with any of the KLF, Oct, Mycand/or Sox polypeptide.

B. Induction of CiPSCs Via Specific Selection of Conditions for XEN-LikeState Bias

Reprogramming non-pluripotent cells via a XEN-like state bias includesthe steps of (a) contacting the cell to be reprogrammed with a firstcocktail of CIPs (XEN-cocktail) for a sufficient period of time to biasthe cells into a XEN-like state, thus generating a subpopulationXEN-like cells; (b) contacting the population of XEN-like cells for asufficient period of time to reprogram the cells into a chemicallyinduced pluripotent stem cell (CiPSC) with a second cocktail of CIPS(XEN-CiPSC cocktail) and (c) culturing the cells in 2i-medium. The cellsare preferably replated during step (a) at a density of about50,000-100,000 cells per well in a 6-well plate.

In a preferred embodiment, cells to be reprogrammed are culturedinitially in a reprogramming medium containing the CIPs for a totalperiod preferably between 26-30 days. The cells are then cultured in2i-medium for more than 4 days. The cells are cultured in 2i-medium frompreferably, between 10-14 days (FIG. 19A) In a preferred embodiment, theVC6TF cocktail is present the entire time before the use of 2i-medium.AM 580 should be used in step (a), and is preferably present the entiretime before the use of 2i-medium. EPZ004777 is preferably added in step(a) and may be present the entire time before the use of the 2i-medium.Accordingly, a preferred XEN-cocktail for biasing/priming cells to bereprogrammed into a XEN-like state is VC6TFAE. 5-azacytidine and shouldbe added in step (b) following treatment with the preferred VC6TFAEcocktail at the late stage of reprogramming (for example, day 16 today28). SGC 0946 is preferably not included in the reprogramming mediumin step (a) for biasing cells into a XEN-like state, but is preferablyincluded in step (b) for programming XEN-like cells into caps.Accordingly, a preferred cocktail for step (b) (XEN-CiPSC-cocktail isVC6TFZASD. In some preferred embodiments, the concentration of forskolinin the XEN-cocktail is different from its concentration in the XEN-CiPSCcocktail (5:1). The XEN-CiPSC-cocktail should be changed into 2i-mediumbetween day 26 to day 30, and preferably day 28. The 2i-mediumpreferably additionally includes N2B27. N2B27-2i medium (500 mL)includes the following: 240 ml DMEM/F12 (Invitrogen), 240 ml Neurobasal(Invitrogen), 5 ml N2 supplement (Invitrogen), 10 ml B27 supplement(Invitrogen), 2 mM GlutaMAX™-I (Invitrogen), 1% nonessential amino acids(Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 1%penicillin-streptomycin (Invitrogen), 3 μM CHIR99021, 1 μM PD0325901 and1,000 U/ml LIF.

Inducing CiPSCs via XEN-like state bias increases the efficiency ofreprogramming. For example, the number of colonies obtained cells to beprogrammed are first biased towards a XEN-LIKE state from the samestarting cell population is increased and/or the length of time it takesto obtain the same number of colonies is reduced when compared to achemical reprogramming method that does not selectively bias the cellsto be programming towards a XEN-like state.

C. Isolation of CiPSCs

Media that can maintain the undifferentiated state and pluripotency ofES cells or induce differentiation are known in this field.Differentiation and proliferation abilities of isolated inducedpluripotent stem cells can be easily confirmed by those skilled in theart by using confirmation means widely applied to ES cells.

A substantially purified population of CiPSCs can be obtained, forexample, by extraction (e.g., via density gradient centrifugation and/orflow cytometry) from a culture source. Purity can be measured by anyappropriate method. The pluripotent cells can be 99%-100% purified by,for example, flow cytometry (e.g., FACS analysis). Human inducedpluripotent stem cells can be isolated by, for example, utilizingmolecules (e.g., antibodies, antibody derivatives, ligands or Fc-peptidefusion molecules) that bind to a marker (e.g., a TRA-1-81, a TRA-1-61 ora combination of markers) on the induced pluripotent stem cells andthereby positively selecting cells that bind the molecule (i.e., apositive selection). Other examples of positive selection methodsinclude methods of preferentially promoting the growth of a desired celltype in a mixed population of desired and undesired cell types.Alternatively, by using molecules that bind to markers that are notpresent on the desired cell type, but that are present on an undesiredcell type, the undesired cells containing such markers can be removedfrom the desired cells (i.e., a negative selection). Other negativeselection methods include preferentially killing or inhibiting thegrowth of an undesired cell type in a mixed population of desired andundesired cell types. Accordingly, by using negative selection, positiveselection, or a combination thereof, an enriched population of stem cellcan be made.

Procedures for separation may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents joined to a monoclonal antibody, or such agents used inconjunction with a monoclonal antibody, e.g., complement and cytotoxins,and “panning” with antibody attached to a solid matrix (e.g., plate), orother convenient technique. Techniques providing accurate separationinclude fluorescence activated cell sorters, which can have varyingdegrees of sophistication, e.g., a plurality of color channels, lowangle and obtuse light scattering detecting channels, and impedancechannels. Antibodies may be conjugated with markers, such as magneticbeads, which allow for direct separation, biotin, which can be removedwith avidin or streptavidin bound to a support, or fluorochromes, whichcan be used with a fluorescence activated cell sorter, to allow for easeof separation of the particular cell type. Any technique may be employedwhich is not unduly detrimental to the viability of the inducedpluripotent stem cells. In one embodiment, the cells are incubated withan antibody against a marker (e.g., a TRA-1-81 antibody) and the cellsthat stain positive for the marker are manually selected andsubcultured.

Combinations of enrichment methods may be used to improve the time orefficiency of purification or enrichment. For example, after anenrichment step to remove cells having markers that are not indicativeof the cell type of interest, the cells may be further separated orenriched by a fluorescence activated cell sorter (FACS) or othermethodology having high specificity. Multi-color analyses may beemployed with a FACS. The cells may be separated on the basis of thelevel of staining for a particular antigen or lack thereof.Fluorochromes may be used to label antibodies specific for a particularantigen. Such fluorochromes include phycobiliproteins, e.g.,phycoerythrin and allophycocyanins, fluorescein, and Texas red.

Any cell type-specific markers can be used to select for or against aparticular cell type. Induced stem cell markers useful for enrichmentcomprise expressed markers such as TRA-1-81 and loss of markers (e.g.,GFP) associated with a retroviral vector or other exogenous vector.

C. Culture and Preservation of CiPSCs (and their Progeny)

The CiPSCs can be expanded in culture and stored for later retrieval anduse. Once a culture of cells or a mixed culture of stem cells isestablished, the population of cells is mitotically expanded in vitro bypassage to fresh medium as cell density dictates under conditionsconducive to cell proliferation, with or without tissue formation. Suchculturing methods can include, for example, passaging the cells inculture medium lacking particular growth factors that inducedifferentiation (e.g., IGF, EGF, FGF, VEGF, and/or other growth factor).Cultured cells can be transferred to fresh medium when sufficient celldensity is reached. Some stem cell types do not demonstrate typicalcontact inhibition-apoptosis or they become quiescent when density ismaximum. Accordingly, appropriate passaging techniques can be used toreduce contact inhibition and quiescence.

Cells can be cryopreserved for storage according to known methods, suchas those described in Doyle et al., (eds.), 1995, Cell & Tissue Culture:Laboratory Procedures, John Wiley & Sons, Chichester. For example, cellsmay be suspended in a “freeze medium” such as culture medium containing15-20% fetal bovine serum (FBS) and 10% dimethylsulfoxide (DMSO), withor without 5-10% glycerol, at a density, for example, of about 4-10×10⁶cells/ml. The cells are dispensed into glass or plastic vials which arethen sealed and transferred to a freezing chamber of a programmable orpassive freezer. The optimal rate of freezing may be determinedempirically. For example, a freezing program that gives a change intemperature of −1° C./min through the heat of fusion may be used. Oncevials containing the cells have reached −80° C., they are transferred toa liquid nitrogen storage area. Cryopreserved cells can be stored for aperiod of years.

IV. Methods of Use

Identification of a readily available source of stem cells that can giverise to a desired cell type or morphology is important for therapeutictreatments, tissue engineering and research. The availability of stemcells would be extremely useful in transplantation, tissue engineering,regulation of angiogenesis, vasculogenesis, and cell replacement or celltherapies as well as the prevention of certain diseases. Such stem cellscan also be used to introduce a gene into a subject as part of a genetherapy regimen.

A. Providing Differentiated Somatic Cells (Re-Differentiated Cells)

Once established, a culture of stem cells may be used to produce progenycells, for example, fibroblasts capable of producing new tissue. TheCiPSCs can be induced to differentiate into cells from any of the threegerm layers, for example, skin and hair cells including epithelialcells, keratinocytes, melanocytes, adipocytes, cells forming bone,muscle and connective tissue such as myocytes, chondrocytes, osteocytes,alveolar cells, parenchymal cells such as hepatocytes, renal cells,adrenal cells, and islet cells, blood cells, retinal cells (and othercells involved in sensory perception, such as those that form hair cellsin the ear or taste buds on the tongue), and nervous tissue includingnerves.

In one embodiment, the CiPSCs are induced to differentiate into cells ofectodermal origin by exposing the cells to an “ectodermaldifferentiating” media. In another embodiment the CiPSCs are induced todifferentiate into cells of mesodermal origin by exposing the cells to“mesodermal differentiating media”. In still another embodiment, theCiPSCs are induced to differentiate into cells of endodermal origin byexposing the cells to “endodermal media”. Components of “endodermal”,“mesodermal” and “ectodermal” media are known to one of skill in theart. Known cell surface markers can be used to verify that the cells areindeed differentiating into cells of the lineage of the correspondingcell culture medium. The most commonly accepted markers to confirmdifferentiation of the three germ layers are the expression of alphafetal protein for endodermal cells, alpha smooth muscle actin formesoderm, and Beta-III tubulin for ectoderm, all of which are normallyexpressed very early in the development of these tissues.

Differentiation of stem cells to fibroblasts or other cell types,followed by the production of tissue therefrom, can be triggered byspecific exogenous growth factors or by changing the culture conditions(e.g., the density) of a stem cell culture. Methods for inducingdifferentiation of cells into a cell of a desired cell type are known inthe art. For example, CiPSCs can be induced to differentiate by adding asubstance (e.g., a growth factor, enzyme, hormone, or other signalingmolecule) to the cell's environment. Examples of factors that can beused to induce differentiation include erythropoietin, colonystimulating factors, e.g., GM-CSF, G-CSF, or M-CSF, interleukins, e.g.,IL-1, -2, -3, -4, -5, -6, -7, -8, Leukemia Inhibitory Factory (LIF), orSteel Factor (Stl), coculture with tissue committed cells, or otherlineage committed cells types to induce the stem cells into becomingcommitted to a particular lineage.

The redifferentiated cells can be can be expanded in culture and storedfor later retrieval and use.

B. Cell Therapy

Therapeutic uses of the induced pluripotent stem cells includetransplanting the induced pluripotent stem cells, stem cell populations,or progeny thereof into individuals to treat a variety of pathologicalstates including diseases and disorders resulting from cancers, wounds,neoplasms, injury, viral infections, diabetes and the like. Treatmentmay entail the use of the cells to produce new tissue, and the use ofthe tissue thus produced, according to any method presently known in theart or to be developed in the future. The cells may be implanted,injected or otherwise administered directly to the site of tissue damageso that they will produce new tissue in vivo. In one embodiment,administration includes the administration of genetically modifiedCiPSCs or their progeny.

In a preferred embodiment, the CiPSCs are obtained from autologous cellsi.e., the donor cells are autologous. However, the cells can be obtainedfrom heterologous cells. In one embodiment, the donor cells are obtainedfrom a donor genetically related to the recipient. In anotherembodiment, donor cells are obtained from a donor genetically un-relatedto the recipient.

If the human CiPSCs are derived from a heterologous(non-autologous/allogenic) source compared to the recipient subject,concomitant immunosuppression therapy is typically administered, e.g.,administration of the immunosuppressive agent cyclosporine or FK506.However, due to the immature state of the human induced pluripotent stemcells such immunosuppressive therapy may not be required. Accordingly,in one embodiment, the human induced pluripotent stem cells can beadministered to a recipient in the absence of immunomodulatory (e.g.,immunsuppressive) therapy. Alternatively, the cells can be encapsulatedin a membrane, which permits exchange of fluids but prevents cell/cellcontact. Transplantation of microencapsulated cells is known in the art,e.g., Balladur et al., Surgery, 117:189-94, 1995; and Dixit et al., CellTransplantation 1:275-79 (1992).

(i) Diabetes

Diabetes mellitus (DM) is a group of metabolic diseases where thesubject has high blood sugar, either because the pancreas does notproduce enough insulin, or, because cells do not respond to insulin thatis produced.

A promising replacement for insulin therapy is provision of islet cellsto the patient in need of insulin. Shapiro et al., N Engl J Med.,343(4):230-8 (2000) have demonstrated that transplantation of betacells/islets provides therapy for patients with diabetes. Althoughnumerous insulin types are commercially available, these formulationsare provided as injectables. The human induced pluripotent stem cellsprovide an alternative source of islet cells to prevent or treatdiabetes. For example, induced pluripotent stem cells can be isolatedand differentiated to a pancreatic cell type and delivered to a subject.Alternatively, the induced pluripotent stem cells can be delivered tothe pancreas of the subject and differentiated to islet cells in vivo.Accordingly, the cells are useful for transplantation in order toprevent or treat the occurrence of diabetes. Methods for reducinginflammation after cytokine exposure without affecting the viability andpotency of pancreatic islet cells are disclosed for example in U.S. Pat.No. 8,637,494 to Naziruddin, et al.

(ii) Neurodegenerative Disorders

Neurodegenerative disorders are characterized by conditions involvingthe deterioration of neurons as a result of disease, hereditaryconditions or injury, such as traumatic or ischemic spinal cord or braininjury. Neurodegenerative conditions include any disease or disorder orsymptoms or causes or effects thereof involving the damage ordeterioration of neurons. Neurodegenerative conditions can include, butare not limited to, Alexander Disease, Alper's Disease, AlzheimerDisease, Amyotrophic Lateral Sclerosis, Ataxia Telangiectasia, CanavanDisease, Cockayne Syndrome, Corticobasal Degeneration, Creutzfeldt-JakobDisease, Huntington Disease, Kennedy's Disease, Krabbe Disease, LewyBody Dementia, Machado-Joseph Disease, Multiple Sclerosis, ParkinsonDisease, Pelizaeus-Merzbacher Disease, Niemann-Pick's Disease, PrimaryLateral Sclerosis, Refsum's Disease, Sandhoff Disease, Schilder'sDisease, Steele-Richardson-Olszewski Disease, Tabes Dorsalis or anyother condition associated with damaged neurons. Other neurodegenerativeconditions can include or be caused by traumatic spinal cord injury,ischemic spinal cord injury, stroke, traumatic brain injury, andhereditary conditions.

In particular, the disclosed methods include transplanting into asubject in need thereof NSCs, neural progenitors, or neural precursorsthat have been expanded in vitro such that the cells can ameliorate theneurodegenerative condition. Transplantation of the expanded neural stemcells can be used to improve ambulatory function in a subject sufferingfrom various forms of myelopathy with symptoms of spasticity, rigidity,seizures, paralysis or any other hyperactivity of muscles. Methods forexpanding and transplanting neural cells and neural progenitor cells forthe treatment of different neurodegenerative conditions is disclosed forexample, in U.S. Pat. No. 8,236,299 to Johe, et. al.

(iii) Cancer Therapy

Therapeutic uses of the CiPSCs and their progeny include transplantingthe induced pluripotent stem cells, stem cell populations, or progenythereof into individuals to treat and/or ameliorate the symptomsassociated with cancer. For example, in one embodiment, the CiPSCs canbe administered to cancer patients who have undergone chemotherapy thathas killed, reduced, or damaged cells of a subject. In a typical stemcell transplant for cancer, very high doses of chemotherapy are used,often along with radiation therapy, to try to destroy all the cancercells. This treatment also kills the stem cells in the bone marrow. Soonafter treatment, stem cells are given to replace those that weredestroyed.

In another embodiment, the CiPSCs can be transfected or transformed (inaddition to the de-differentiation factors) with at least one additionaltherapeutic factor. For example, once CiPSCs are isolated, the cells maybe transformed with a polynucleotide encoding a therapeutic polypeptideand then implanted or administered to a subject, or may bedifferentiated to a desired cell type and implanted and delivered to thesubject. Under such conditions the polynucleotide is expressed withinthe subject for delivery of the polypeptide product.

(iii) Tissue Engineering

CiPSCs and their progeny can be used to make tissue engineeredconstructions, using methods known in the art. Tissue engineeredconstructs may be used for a variety of purposes including as prostheticdevices for the repair or replacement of damaged organs or tissues. Theymay also serve as in vivo delivery systems for proteins or othermolecules secreted by the cells of the construct or as drug deliverysystems in general, Tissue engineered constructs also find use as invitro models of tissue function or as models for testing the effects ofvarious treatments or pharmaceuticals. The most commonly usedbiomaterial scaffolds for transplantation of stem cells are reviewed inthe most commonly used biomaterial scaffolds for transplantation of stemcells is reviewed in Willerth, S. M. and Sakiyama-Elbert, S. E.,Combining stem cells and biomaterial scaffolds for constructing tissuesand cell delivery (Jul. 9, 2008), StemBook, ed. The Stem Cell ResearchCommunity, StemBook. Tissue engineering technology frequently involvesselection of an appropriate culture substrate to sustain and promotetissue growth. In general, these substrates should be three-dimensionaland should be processable to form scaffolds of a desired shape for thetissue of interest.

U.S. Pat. No. 6,962,814 generally discloses method for producing tissueengineered constructs and engineered native tissue. With respect tospecific examples, U.S. Pat. No. 7,914,579 to Vacanti, et al., disclosestissue engineered ligaments and tendons. U.S. Pat. No. 5,716,404discloses methods and compositions for reconstruction or augmentation ofbreast tissue using dissociated muscle cells implanted in combinationwith a polymeric matrix. U.S. Pat. No. 8,728,495 discloses repair ofcartilage using autologous dermal fibroblasts. U.S. Publishedapplication No. 20090029322 by Duailibi, et al., discloses the use ofstem cells to form dental tissue for use in making tooth substitute.U.S. Published application No. 2006/0019326 discloses cell-seedtissue-engineered polymers for treatment of intracranial aneurysms. U.S.Published application No. 2007/0059293 by Atala discloses thetissue-engineered constructs (and method for making such constructs)that can be used to replace damaged organs for example kidney, heart,liver, spleen, pancreas, bladder, ureter and urethra.

(ii) Cells Produced from CiPSCs (Progeny)

The CiPSCs can be induced to differentiate into cells from any of thethree germ layers, for example, skin and hair cells including epithelialcells, keratinocytes, melanocytes, adipocytes, cells forming bone,muscle and connective tissue such as myocytes, chondrocytes, osteocytes,alveolar cells, parenchymal cells such as hepatocytes, renal cells,adrenal cells, and islet cells (e.g., alpha cells, delta cells, PPcells, and beta cells), blood cells (e.g., leukocytes, erythrocytes,macrophages, and lymphocytes), retinal cells (and other cells involvedin sensory perception, such as those that form hair cells in the ear ortaste buds on the tongue), and nervous tissue including nerves.

(iii) Therapeutic Compositions

The CiPSCs can be formulated for administration, delivery or contactingwith a subject, tissue or cell to promote de-differentiation in vivo orin vitro/ex vivo. Additional factors, such as growth factors, otherfactors that induce differentiation or dedifferentiation, secretionproducts, immunomodulators, anti-inflammatory agents, regressionfactors, biologically active compounds that promote innervation,vascularization or enhance the lymphatic network, and drugs, can beincorporated.

The induced pluripotent cells can be administered to a patient by way ofa composition that includes a population of CiPSCs or CiPSC progenyalone or on or in a carrier or support structure. In many embodiments,no carrier will be required. The cells can be administered by injectiononto or into the site where the cells are required. In these cases, thecells will typically have been washed to remove cell culture media andwill be suspended in a physiological buffer.

In other embodiments, the cells are provided with or incorporated ontoor into a support structure. Support structures may be meshes, solidsupports, scaffolds, tubes, porous structures, and/or a hydrogel. Thesupport structures may be biodegradable or non-biodegradable, in wholeor in part. The support may be formed of a natural or synthetic polymer,metal such as titanium, bone or hydroxyapatite, or a ceramic. Naturalpolymers include collagen, hyaluronic acid, polysaccharides, andglycosaminoglycans. Synthetic polymers include polyhydroxyacids such aspolylactic acid, polyglycolic acid, and copolymers thereof,polyhydroxyalkanoates such as polyhydroxybutyrate, polyorthoesters,polyanhydrides, polyurethanes, polycarbonates, and polyesters. These maybe in for the form of implants, tubes, meshes, or hydrogels.

Solid Supports

The support structure may be a loose woven or non-woven mesh, where thecells are seeded in and onto the mesh. The structure may include solidstructural supports. The support may be a tube, for example, a neuraltube for regrowth of neural axons. The support may be a stent or valve.The support may be a joint prosthetic such as a knee or hip, or partthereof, that has a porous interface allowing ingrowth of cells and/orseeding of cells into the porous structure. Many other types of supportstructures are also possible. For example, the support structure can beformed from sponges, foams, corals, or biocompatible inorganicstructures having internal pores, or mesh sheets of interwoven polymerfibers. These support structures can be prepared using known methods.

The support structure may be a permeable structure having pore-likecavities or interstices that shape and support the hydrogel-cellmixture. For example, the support structure can be a porous polymermesh, a natural or synthetic sponge, or a support structure formed ofmetal or a material such as bone or hydroxyapatite. The porosity of thesupport structure should be such that nutrients can diffuse into thestructure, thereby effectively reaching the cells inside, and wasteproducts produced by the cells can diffuse out of the structure

The support structure can be shaped to conform to the space in which newtissue is desired. For example, the support structure can be shaped toconform to the shape of an area of the skin that has been burned or theportion of cartilage or bone that has been lost. Depending on thematerial from which it is made, the support structure can be shaped bycutting, molding, casting, or any other method that produces a desiredshape. The support can be shaped either before or after the supportstructure is seeded with cells or is filled with a hydrogel-cellmixture, as described below.

An example of a suitable polymer is polyglactin, which is a 90:10copolymer of glycolide and lactide, and is manufactured as VICRYL™braided absorbable suture (Ethicon Co., Somerville, N.J.). Polymerfibers (such as VICRYL™), can be woven or compressed into a felt-likepolymer sheet, which can then be cut into any desired shape.Alternatively, the polymer fibers can be compressed together in a moldthat casts them into the shape desired for the support structure. Insome cases, additional polymer can be added to the polymer fibers asthey are molded to revise or impart additional structure to the fibermesh. For example, a polylactic acid solution can be added to this sheetof polyglycolic fiber mesh, and the combination can be molded togetherto form a porous support structure. The polylactic acid binds thecrosslinks of the polyglycolic acid fibers, thereby coating theseindividual fibers and fixing the shape of the molded fibers. Thepolylactic acid also fills in the spaces between the fibers. Thus,porosity can be varied according to the amount of polylactic acidintroduced into the support. The pressure required to mold the fibermesh into a desirable shape can be quite moderate. All that is requiredis that the fibers are held in place long enough for the binding andcoating action of polylactic acid to take effect.

Alternatively, or in addition, the support structure can include othertypes of polymer fibers or polymer structures produced by techniquesknown in the art. For example, thin polymer films can be obtained byevaporating solvent from a polymer solution. These films can be castinto a desired shaped if the polymer solution is evaporated from a moldhaving the relief pattern of the desired shape. Polymer gels can also bemolded into thin, permeable polymer structures using compression moldingtechniques known in the art.

Hydrogels

In another embodiment, the cells are mixed with a hydrogel to form acell-hydrogel mixture. Hydrogels may be administered by injection orcatheter, or at the time of implantation of other support structures.Crosslinking may occur prior to, during, or after administration.

V. Kits

Kits are provided which include the chemical inducers of pluripotency(CIP) disclosed herein. The CIPs are as described above. These may be ina form having defined concentrations to facilitate addition to cellculture media to produce a desired concentration. The kit may includedirections providing desired concentration ranges and times ofadministration based on the types of cells to be induced. The kit mayalso include cell culture media which is pre-mixed with the CIPs forculture of cells to induce pluripotency.

The present invention will be further understood by reference to thefollowing non-limiting examples.

Examples Materials and Methods

Mice

The mouse strains C57BL/6 J-Tg(GOFGFP)11Imeg/Rbrc (OG), C57BL/6NCrlVr(C57), ICR and 129S2/SvPasCrlVr (129) were purchased as described by Li,Cell Res., 21:196-204 (2011). The OG mice were mated with other strainsto generate offspring carrying Oct4 promoter-driven GFP. Mouse strainsincluding ICR, C57×129, OG×ICR, OG×129 and OG×C57 were used to isolateprimary mouse embryonic fibroblasts (MEFs), mouse neonatal fibroblasts(MNFs), mouse adult fibroblasts (MAFs) and adipose-derived stem cells(ADSCs). These cells were used for CiPSC induction. The neonatal miceused were 2-3 days old and the adult mice used were 7 weeks old. TheTet-On POU5F1 mouse strain B6;129t(ROSA)26Sortm1(rtTA*M2)JaeCol1a1tm2(tetO-Pou5f1)Jae/J was purchased from Jackson Laboratory(Hochedlinger, et al., Cell, 121:465-477 (2005)) and used only forreprogramming with Oct4 plus VC6 T. Animal experiments were performedaccording to the Animal Protection Guidelines of Peking University,China.

Cell Culture

Primary MEFs were isolated as described by Takahashi, et al., Cell,126:663-676 (2006)), with careful attention to the removal of thegenital ridges. MNFs from skin, MAFs from lungs and ADSCs from inguinalfat pads were isolated as described by Lichti, et al., Nat. Protoc.3:799-810 (2008); Seluanov, et al., J. Vis. Exp. 2010:2033 (2010); Tat,et al., Cell Transplant. 19:525-536 (2010) and McQualter, et al., StemCells, 27:623-633 (2009).

MEFs, MNFs, MAFs and ADSCs were cultured in DMEM/High Glucose (Hyclone)containing 10% fetal bovine serum (Hyclone). The cells used inreprogramming were from passages 1 to 5.

Mouse ESCs (R1 and TT2), iPSCs and CiPSCs were maintained on feederlayers of mitomycin C-treated MEFs in ESC culture medium (KnockOut DMEM(Invitrogen) containing 10% knockout serum replacement (Invitrogen), 10%fetal bovine serum (Hyclone), 2 mM GlutaMAX™-I (Invitrogen), 1%nonessential amino acids (Invitrogen), 0.1 mM 2-mercaptoethanol(Invitrogen), 1% penicillin-streptomycin (Invitrogen) and 1,000 U/mlleukemia inhibitory factor (LIF, Millipore)) or 2i-medium (ESC culturemedium supplemented with 2i (3 μM CHIR99021 and 1 μM PD0325901)). Themedium was changed daily. ESCs, iPSCs and CiPSCs were passaged bytrypsin-EDTA (Invitrogen). For CiPSC induction, LIF-free ESC culturemedium supplemented with 20-100 ng/ml bFGF (Origene) was used as thechemical reprogramming medium.

Fetal small intestinal epithelial cells were isolated from mouseembryonic small intestine at embryonic 13.5 day as previously described(Li et al, 2011), and cultured in Knockout™ DMEM (Invitrogen),supplemented with 10% fetal bovine serum (FBS; Pan-Biotech), 10%knockout serum replacement (KSR), 1% non-essential amino acids (NEAA), 2mM GlutaMAX™-I (GlutaMAX), 10 U penicillin-streptomycin (PS), and 55 μMβ-Mercaptoethanol (β-me) (all from Invitrogen).

Fetal neural stem cells (NSCs) were isolated from mouse forebrain atembryonic day 13.5 as previously described (Fischer et al., 2011), andpostnatal NSCs were isolated from the subventricular zone of thepostnatal mouse brain (Fischer et al., 2011: Guo et al., 2012). NSCswere cultured in NSC culture medium (DMEM/F-12 (1:1), DF12) containingN2 and B27 supplements, 1% NEAA, 2 mM GlutaMAX, 10 U PS, 55 μM β-me (allfrom Invitrogen), 25 ng/mL basic fibroblast growth factor (bFGF)(Origene), and 20 ng/mL epidermal growth factor (EGF) (R&D)), andpassaged by accutase (Millipore) every 4-5 days. NSCs were single-cellsuspended and formed neurospheres for 2-3 days.

Mouse ESCs (R1) were maintained on feeder layers of mitomycin C-treatedMEFs in 2i-medium plus LIF (Knockout™ DMEM containing 10% KSR, 10% FBS,2 mM GlutaMAX, 1% NEAA, 55 μM β-me, 10 U PS (all from Invitrogen), 3 μMCHIR99021 (CHIR), 1 μM PD0325901 (PD03) and 10 ng/mL mouse leukemiainhibitory factor (mLIF; Millipore)). The medium was changed daily. ESCsand CiPSCs were passaged by trypsin-EDTA (Invitrogen).

For CiPSC induction, ESC culture medium without CHIR, PD03 and LIFsupplemented with bFGF and Vc was used as chemical reprogramming medium.At the 2i-medium stage, the basal culture medium was DMEM/F12 plus N2and B27 supplements (N2B27-2iL medium).

XEN Cell Derivation and Culture

Traditional eXEN (embryo-derived XEN) cell lines (gift from Dr.Rossant's laboratory) were cultured as previously described (Kunath etal., 2005). Briefly, eXEN cells were seeded on MEF feeders with RPMI1640(Invitrogen) containing 20% fetal bovine serum, 2 mM GlutaMAX™-I(Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM2-mercaptoethanol (Invitrogen), 1% penicillin-streptomycin (Invitrogen),and 1% sodium pyruvate (Invitrogen; RPMI medium). For most experiments,eXEN cells were cultured in a feeder-free system, on gelatin (0.1%porcine skin gelatin, Sigma)-coated plates supplemented with 70%MEF-conditioned medium (RPMI-CM). Alternatively, eXEN could be culturedlong-term in stage 1 medium (with VC6TFAE or VC6TF; with 20 ng/ml bFGF)on gelatin-coated plates (eXEN in VC6TF). Cells were routinely fed every2 days and passaged at 1:20 to 1:30 every 3 days. eXEN were also deriveddirectly from E3.5 blastocysts using stage 1 medium (with VC6TFAE orVC6TF; with 20 ng/ml bFGF; CeXEN). In brief, E3.5 blastocysts wereplated on MEF feeders in stage 1 medium. The medium was changed every 2days. Approximately 4-7 days (depending on the size of outgrowth) later,outgrowths could be disaggregated 1:1 to 1:2 onto new feeder cells. Themedium was changed every 2 days, and XEN cells could grow to confluencein approximately 4-7 days. Then, CeXEN cells were routinely cultured instage 1 medium and passaged 1:20-1:30 every 3 days. Three CeXEN celllines were derived in this manner and were expanded long-term for morethan 25 passages.

Small-Molecule Compounds and Libraries

The small-molecule compounds used in this study were purchased orsynthesized as described in Table 1D. The concentration of compounds isshown in Table 1D. Small-molecule libraries used for the screen werepurchased or generated in-house as described in Table 1C

TABLE 1C Small molecule libraries used in reprograming Number of small-Library Source molecule compounds BBP-2080NPs library BioBioPha 2,080The Spectrum Collection MicroSource 2,000 Discovery Systems SigmaLOPAC ®^(,1280) Sigma 1,280 Prestwick Chemical Prestwick Chemical 1,200Library ® Tocriscreen ™ Total Tocris 1,120 US Drug CollectionMicroSource 1,040 Discovery Systems ICCB Known Enzo 480 BioactivesLibrary Protein Kinase Inhibitor Millipore 324 Library I, II, IIIStemSelect Small Calbiochem 303 Molecule Regulators Nuclear ReceptorEnzo 76 Ligand Library Selected Small Molecules* Our lab 88 *Thislibrary was generated in-house, including 88 selected small moleculesrelated to pluripotency, reprogramming or epigenetic modification

TABLE 1D Small-molecule compounds tested in reprogramming Concen- Abbre-tration Molecular Full Name viation (μM) Source Weight StructureValproic acid sodium salt VPA, V 500 Sigma, cat. no. P4543 166.19

CHIR99021 CHIR, C 10-20* Synthesized by WUXI APPTEC 465.34

616452 6 5-10 Synthesized by WUXI APPTEC 287.12

Tranylcypro- mine Tranyl, T 5-10 Enzo, cat. no. BML- EI217-0005 182.23

Forskolin FSK, F  10-50** Enzo, cat. no. BML- CN100-0100 410.50

3- deazaneplan- ocin A DZNep, Z 0.05-0.1  Synthesized by WUXI APPTEC262.26

2-Methyl-5- hydroxytryp- tamine hydro- chloride 2-Me- 5HT, M  5Synthesized by WUXI APPTEC 233.99

D4476 D  5 Synthesized by WUXI APPTEC 398.41

PD0325901  1 Synthesized by WUXI APPTEC 482.00

Adenosine, periodate oxidized Adox  10 Santa Cruz, cat no. sc- 214510265.23

IBMX  50 Tocris, cat. no. 2845 222.24

Dibutyryl- cAMP DBcAMP  50 Santa Cruz, cat. no. sc- 201567 491.37

2′,5′- Dideoxy- adenosine 2′5′ddAdo 5-20 Santa Cruz, cat. no. sc- 201562235.2 

Prosta- glandin E2 PGE2, P  5 Cayman, cat. no. 14010 352.46

Rolipram  10 Tocris, cat. no. 0905 275.35

Sodium butyrate NaB, B  20 Sigma, cat. no. B5887 110.09

SRT1720 S  1 Selleck, cat. no. S1129 506.02

UNC0638 UNC, U  0.5 Tocris, cat. no. 4343 509.73

Ionomycin  4 Calbiochem, cat. no. 407952 747.06

BIX-01294  1 Stemgent, cat. no. 04- 0002 490.64

(−)- Neplanocin A Nep A  1 Cayman, cat. no. 10584 263.25

3- Deaza- adenosine DZA  10 Cayman, cat. no. 90000785 266.25

Budesonide Bude  5 Tocris cat. no. 2671 430.53

RG108 R 20-40  Tocris, cat. no. 3295 334.33

5- Azacytidine 5-aza-C  5 Tocris, cat. no. 3842 244.2 

Adenine  2 Calbiochem, cat. no. 1152 135.13

Adenosine  2 Calbiochem, cat. no. 1160CBC 267.24

SF1670  1 Cellagen Technology, cat. no. C7316-2s 307.34

DY131  5 Tocris, cat. no. 2266 311.38

Nimesulide  2 Tocris, cat. no. 2470 308.31

Resveratrol  3 Tocris, cat. no. 1418 228.25

TTNPB N  1 Tocris, cat. no. 0761 348.48

Note: *For CiPSCs induction from MEFs, the concentration of CHIR99021was 10 μM. For CiPSCs induction from MNFs, MAFs or ADSCs, and CiPSCsinduction without replating, the concentration of CHIR99021 was elevatedto 20 μM during day 0-12. **For CiPSCs induction from MEFs, theconcentration of Forskolin was 10 μM. For CiPSCs induction from MNFs,MAFs or ADSCs, the concentration of Forskolin was elevated to 50 μMduring day 0-12.

Plasmid Construction and Lentivirus Production

The pLL3.7-ΔU6 vector was described by (McQualter, et al., Stem Cells,27:623-633 (2009). Mouse Sall4 was amplified from ESCs (TT2) by RT-PCR,cloned into the pEASY-Blunt vector (TransGen Biotech), confirmed bysequencing and then introduced into the XhoI/EcoRI sites of pLL3.7-ΔU6.The primers are listed in Table 2.

TABLE 2 Primer sets for PCR reactions Genes Forward (5′ to 3′)Reverse (5′ to 3′) For plasmid construction Sall4 ACTCGAGCCACCATGTCGAGGCGCAATTGTTAGCTGACAGCAAT GCAAGCAGGCGAA (SEQ ID NO:CTTATTTTCCTCC (SEQ ID NO: 2) 1) For qRT-PCR Sox2 CGGGAAGCGTGTACTTATCCTTGCGGAGTGGAAACTTTTGTCC (SEQ ID NO: 3) (SEQ ID NO: 4) Klf4TTGCGGTAGTGCCTGGTCAGTT CTATGCAGGCTGTGGCAAAACC (SEQ ID NO: 5)(SEQ ID NO: 6) Oct4 CAGGGCTTTCATGTCCTGG AGTTGGCGTGGAGACTTTGC(SEQ ID NO: 7) (SEQ ID NO: 8) Gata4 GAGCTGGCCTGCGATGTCTGAGAAACGGAAGCCCAAGAACCTGA TG (SEQ ID NO: 9) AT (SEQ ID NO: 10) Gata6TGAGGTGGTCGCTTGTGTAG ATGGCGTAGAAATGCTGAGG (SEQ ID NO: 11)(SEQ ID NO: 12) Sox17 GTCAACGCCTTCCAAGACTTG GTAAAGGTGAAAGGCGAGGTG(SEQ ID NO: 13) (SEQ ID NO: 14) Esrrb GTGGCTGAGGGCATCAATGAACCGAATGTCGTCCGAAGAC (SEQ ID NO: 15) (SEQ ID NO: 16) Sall4TGGCAGACGAGAAGTTCTTTC TCCAACATTTATCCGAGCACAG (SEQ ID NO: 17)(SEQ ID NO: 18) Lin28a CCGCAGTTGTAGCACCTGTCT GAAGAACATGCAGAAGCGAAG(SEQ ID NO: 19) A (SEQ ID NO: 20) Dppa2 GCGTAGCGTAGTCTGTGTTTGTCAACGAGAACCAATCTGAGGA (SEQ ID NO: 21) (SEQ ID NO: 22) NanogAGTTATGGAGCGGAGCAGCAT AGGCCTGGACCGCTCAGT (SEQ (SEQ ID NO: 23) ID NO: 24)Actb CATTGCTGACAGGATGCAGAA TGCTGGAAGGTGGACAGTGAGG GG (SEQ ID NO: 25)(SEQ ID NO: 26) Gadph CATCACTGCCACCCAGAAGACT ATGCCAGTGAGCTTCCCGTTCAGG (SEQ ID NO: 27) (SEQ ID NO: 28) For genomic PCR pLL-Oct4GAAGGATGTGGTCCGAGT (SEQ GCAGCGTATCCACATAGCGT ID NO: 29) (SEQ ID NO: 30)pLL-Sox2 CATGGGTTCGGTGGTCAA (SEQ GCAGCGTATCCACATAGCGT ID NO: 31)(SEQ ID NO: 32) pLL-Klf4 ACCACTGTGACTGGGACG (SEQ GCAGCGTATCCACATAGCGTID NO: 33) (SEQ ID NO: 34) pLL-cMyc TACATCCTGTCCGTCCAAGCGCAGCGTATCCACATAGCGT (SEQ ID NO: 35) (SEQ ID NO: 36) Fu-tet-ACCTCCATAGAAGACACCG TAGCCCCACTCCAACCTG (SEQ hOct4 (SEQ ID NO: 37)ID NO: 38) Fu-tet- ACCTCCATAGAAGACACCG CTCCGACAAAAGTTTCCACTCG hSox2(SEQ ID NO: 39) (SEQ ID NO: 40) Fu-tet- ACCTCCATAGAAGACACCGGAAGAGGAGGCTGACGCT (SEQ hKlf4 (SEQ ID NO: 41) ID NO: 42) Fu-tet-ACCTCCATAGAAGACACCG GGGTCGCAGATGAAACTC (SEQ hcMyc (SEQ ID NO: 43)ID NO: 44) For bisulfite genomic sequencing Oct4 GGAGTGGTTTTAGAAATAATTGTCCAACCCTACTAACCCATCACC (SEQ ID NO: 45) (SEQ ID NO: 46) NanogGATTTTGTAGGTGGGATTAATT ACCAAAAAAACCCACACTCATA GTGAATTT (SEQ ID NO: 47)TCAATATA (SEQ ID NO: 48) For chromatin immunoprecipitation Oct4CTGTAAGGACAGGCCGAGAG CAGGAGGCCTTCATTTTCAA (SEQ ID NO: 49 (SEQ ID NO: 50)Nanog CTATCGCCTTGAGCCGTTG AACTCAGTGTCTAGAAGGAAAG (SEQ ID NO: 51)ATCA (SEQ ID NO: 52) Sox2 TTTATTCAGTTCCCAGTCCAA TTATTCCTATGTGTGAGCAAGA(SEQ ID NO: 53) (SEQ ID NO: 54)For OG (Oct4 promoter-driven GFP) cassette OG AACCACTACCTGAGCACCCACCTCTACAAATGTGGTATG (SEQ ID NO: 55) (SEQ ID NO: 56)The lentiviral vectors containing the other individual reprogrammingfactors (Oct4, Sox2, Klf4 or c-Myc) were described by Zhao, et al., CellStem Cell, 3:475-479 (2008). For tet-on Oct4 systems, Fu-tet-hOct4 andFUdeltaGW-rtTA were the same as described by Li, et al., Cell Res.,21:196-204 (2011); Maherali, et al., Cell Stem Cell, 3:340-345 (2008).Genetic knockdown was carried out using shRNAs (SIGMA MissionR shRNA)according to the manufacturer's protocol. The shRNA sequences are listedin Table 3. Lentivirus production, collection and infection were asdescribed by Zhao, et al., Cell Stem Cell, 3:475-479 (2008).

TABLE 3 Sequences of shRNAs shRNA Sequences (5′-3′) Sall4 shRNCCGGCAGCCCACCTTTGTCAAAGTTCTCGAGAACTTTGACA A 1AAGGTGGGCTGTTTTTG (SEQ ID NO: 57) shRNCCGGGCCCACCTTTGTCAAAGTTGACTCGAGTCAACTTTGA A 2CAAAGGTGGGCTTTTTG (SEQ ID NO: 58) Gata4 shRNCCGGAGCCCAAGAACCTGAATAAATCTCGAGATTTATTCAG A 1GTTCTTGGGCTTTTTTG (SEQ ID NO: 59) shRNCCGGCATCTCCTGTCACTCAGACATCTCGAGATGTCTGAGT A 2GACAGGAGATGTTTTTG (SEQ ID NO: 60) Gata6 shRNCCGGCCACTACCTTATGGCGTAGAACTCGAGTTCTACGCCA A 1TAAGGTAGTGGTTTTTG (SEQ ID NO :61 shRNCCGGCCTCGACCACTTGCTATGAAACTCGAGTTTCATAGCA A 2AGTGGTCGAGGTTTTTG (SEQ ID NO: 62) Sox17 shRNCCGGCCCACAATCACTGTCCAGTTTCTCGAGAAACTGGACA A 1GTGATTGTGGGTTTTTG (SEQ ID NO: 63) shRNCCGGCGCACGGAATTCGAACAGTATCTCGAGATACTGTTCG A 2AATTCCGTGCGTTTTTG (SEQ ID NO: 64) Ezh2 shRNCCGGGCTAGGCTAATTGGGACCAAACTCGAGTTTGGTCCCA A 1ATTAGCCTAGCTTTTTG (SEQ ID NO: 65) shRNCCGGCGGCTCCTCTAACCATGTTTACTCGAGTAAACATGGT A 2TAGAGGAGCCGTTTTTG (SEQ ID NO: 66) Control shRNCCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCT ATCATCTTGT GTTTTTG (SEQ ID NO: 67) control

CiPSC Induction

CiPSC Induction without Selective Priming for XEN-Like Cell Population

The initial cells (MEFs, MNFs, MAFs or ADSCs) were seeded at a densityof 50,000 cells per well of a 6-well plate or 300,000 cells per 100 mmdish. On the next day (day 0), the original medium was replaced withchemical reprogramming medium containing the small-moleculecombinations. The small-molecule combinations-containing medium waschanged every 4 days. On day 12, these cells were washed in PBS anddigested with 0.25% Trypsin-EDTA (Invitrogen) at 37° C. for 3-5 min.After neutralization, the cell clumps were dissociated into single cellsby thorough pipetting. The cells were harvested (300,000-1,000,000 cellsper well of a 6-well plate) and replated at a density of 300,000-500,000cells per well of a 6-well plate in the chemical reprogramming mediumcontaining the small-molecule combinations. DZNep was added to the cellcultures on day 16 or day 20. On day 28-36, the small-moleculecombinations including DZNep were removed. Meanwhile, the chemicalreprogramming medium was replaced with 2i-medium. After another 8-12days, 2i-competent, ESC-like and GFP-positive colonies were counted asprimary CiPSC colonies. For CiPSC induction from wild-type cells withoutOG reporter, 2i-competent and ESC-like colonies were counted as primaryCiPSC colonies. These CiPSC colonies were picked up for expansion andcharacterization. Alternatively, CiPSCs could be induced withoutreplating on day 12.

IEC-CiPSC Induction

The initial IECs were seeded at a density of 100,000 cells per well of a12-well plate. On the next day (day 0), the medium was replaced withchemical reprogramming medium containing the small-molecule cocktail(0.5 mM VPA, 20 μM CHIR, 10 μM 616452, 10 μM Tranylcypromine, 50 μMForskolin, 0.05 μM AM 580) and changed every 4 days. From day 12, 0.01μM DZnep was added into chemical reprogramming medium, and AM 580 iswithdrawn. The medium was replaced with N2B27-2iL medium from day 32.After another 12 days, 2i-competent, ESC-like and OG-positive colonieswere counted as primary CiPSC colonies. The primary CiPSC colonies werepicked up for expansion and further characterization.

NSC-CiPSC Induction

The initial NSCs were seeded at the density of 50,000 cells per well ofa 6-well plate. The original culture medium was replaced by chemicalreprogramming medium containing the small-molecule cocktail (0.5 mM VPA,15 μM CHIR, 5 μM 616452, 10 μM Tranylcypromine, 20 μM Forskolin, 1 μM Ch55, 5 μM EPZ) and changed every 4 days. From day 20, 0.01 μM DZNep wasadded into the chemical reprogramming medium. The medium was replacedwith N2B27-2iL medium from day 40-44. After another 12 days,2i-competent, ESC-like and OG-positive colonies were counted as primaryCiPSC colonies. The primary CiPSC colonies were picked up for expansionand further characterization.

CiPSC Induction Via Selective Priming/Bias for XEN-Like Cell Population

The induction medium was prepared as following: The basal medium ofstage 1 and stage 2 were LIF-free ESC culture medium containing 100ng/ml and 20 ng/ml bFGF (Origene), respectively. The stage 3 medium wasan N2B27-2i medium. The N2B27-2i medium (500 ml) was generated includingthe following: 240 ml DMEM/F12 (Invitrogen), 240 ml Neurobasal(Invitrogen), 5 ml N2 supplement (Invitrogen), 10 ml B27 supplement(Invitrogen), 2 mM GlutaMAX™-I (Invitrogen), 1% nonessential amino acids(Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 1%penicillin-streptomycin (Invitrogen), 3 μM CHIR99021, 1 μM PD0325901 and1,000 U/ml LIF.

MEFs, MNFs or MAFs were plated at 300,000 cells per 100 mm dish, or50,000 cells per well on a 6-well plate. The next day (day 0), theculture was changed into stage 1 medium supplemented with small-moleculecocktail VC6TFAE (0.5 mM VPA, 20 μM CHIR99021, 10 μM 616452, 5 μMTranylcypromine, 50 μM FSK, 0.05 μM AM580 and 5 μM EPZ004777). On day12, the cells were washed in PBS and digested with 0.25% Trypsin-EDTA(Invitrogen) at 37° C. for 2-3 min. After neutralization, the cellclumps were dissociated into single cells by thorough pipetting. Thecells were harvested and then re-plated at 100,000 cells per well of a6-well plate (1:15). During days 12-16, the cells were cultured in stage1 medium supplemented with a modified small-molecule cocktail VC6TFA(0.5 mM VPA, 10 μM CHIR99021, 10 μM 616452, 5 μM Tranylcypromine, 10 μMFSK, 0.05 μM AM580). On day 16, XEN-like epithelial colonies were formedand the culture was changed into stage 2 medium supplemented withsmall-molecule cocktail VC6TFAZDS (0.5 mM VPA, 10 μM CHIR99021, 10 μM616452, 5 μM Tranylcypromine, 10 μM FSK, 0.05 μM AM580, 0.05 μM DZNep,0.5 μM 5-aza-dC and 5 μM SGC0946). On day 28, the culture wastransferred into stage 3 medium. After another 8-12 days, 2i-competent,ESC-like and GFP-positive (if using pOct4-GFP reporter) CiPSC coloniesemerged and were then picked up for expansion and characterization.During CiPSC induction, the medium and small molecules were changedevery 4 days.

Chemical Reprogramming of eXEN Cells

eXEN cells or CeXEN cells were plated at 2,000-10,000 cells per well ofa 12-well plate on MEF feeders. Following the treatment with stage 1medium for 4 days (dispensable for induction), stage 2 medium for 12days and stage 3 medium for another 8-12 days, 2i-competent, ESC-likeCiPSC colonies emerged and were then picked up for expansion andcharacterization. For most experiments, feeder cells were helpful forthe survival of XEN cells, and a relatively low cell density wasbeneficial for CiPSC induction.

Plasmid Construction and Lentivirus Production

Plasmids were constructed as previously described (Zhao et al., 2008).Briefly, SALL4, GATA4 and GATA6 were amplified by qRT-PCR, cloned intothe pEASY-Blunt vector (TransGen Biotech), confirmed by sequencing andthen introduced into the XhoI/EcoRI sites of pLL3.7-ΔU6. The primers arelisted in Table 4.

TABLE 4 Primers for plasmid construction ACCESSION SYMBOL NUMBERSPRIMERS (5′ to 3′) SALL4 NM_020436 TACTCGAGGCCACCATGTCGAGGCGCAAGCAGG(SEQ ID NO: 68) GGGAATTCATCACAAAGCAGCATAGCAACAATC GTG (SEQ ID NO: 69)GATA4 NM_002052 ACCTCGAGGCCACCATGTATCAGAGCTTGGCCA TGGC (SEQ ID NO: 70)GCGAATTCATCATTACGCAGTGATTATGTCCCC GTG (SEQ ID NO: 71) GATA6 NM_005257ATCTCGAGGCCACCATGGCCTTGACTGACGGCG G (SEQ ID NO: 72CTGAATTCATCATCAGGCCAGGGCCAGGGC (SEQ ID NO: 73)

Fu-tet-hSOX2 and FUdeltaGW-rtTA were described previously (Li et al.,2011; Maherali et al., 2008). Genetic knockdown, lentivirus productionand infection were also the same as previously described (Hou et al.,2013).

Immunofluorescence, RT-PCR, Genomic PCR, Teratoma Formation andKaryotype Analysis

Immunofluorescence, RT-PCR, genomic PCR and teratoma formation were allcarried out as described in Hou, et al., Science, 341:651-654 (2013).For immunofluorescence, the primary antibodies included SSEA-1(Millipore, MAB4301), OCT4 (Abcam, ab18976), SOX2 (Santa Cruz,sc-17320), KLF4 (Santa Cruz, sc-20691), REX1 (Santa Cruz, sc-99000),NANOG (R&D, AF2729), UTF1 (Abcam, ab24273), SALL4 (Santa Cruz,sc-166033). Secondary antibodies were Rhodamine-conjugated, includingDonkey Anti Mouse IgG (H+L)(Jackson ImmunoResearch, 715-025-150), DonkeyAnti Goat IgG (H+L) (Jackson ImmunoResearch, 705-025-147), and DonkeyAnti Rabbit IgG (H+L) (Jackson ImmunoResearch, 711-025-152). Primers forRT-PCR were the same as described previously (Li, et al., Cell Res.,21:196-204 (2011)). Primers for genomic PCR are shown in Table 2.Karyotype analyses were performed as reported (Longo, et al., TransgenicRes. 6:321-328 (1997)).

For the imaging analysis, the cells were imaged using the Andor'sRevolution WD spinning disk confocal microscopy system (Andor) orImageXpress Micro XL Widefield High Content Screening System (MolecularDevices).

Real-Time PCR

Total RNA from an entire well of cultured cells was isolated using theRNeasy Plus MiniKit (QIAGEN). For a single colony, RNA was isolatedusing the RNeasy Micro Kit (QIAGEN). RNA was converted to cDNA usingTransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech). PCRwas carried out using Power SYBRR Green PCR Master Mix (AppliedBiosystems) and performed on an ABI Prism 7300 Sequence DetectionSystem. The data were analyzed using the delta-delta Ct method. Theprimers used for real-time PCR are listed in Table 2.

Chimera Construction

Chimeric mice were obtained by the injection of CiPS cells intoblastocysts using a sharp injection needle or into eight-cell embryosusing a XY Clone laser system (Hamilton ThorneBioscience). Forblastocyst injection, 10-15 CiPS cells were injected into the recipientembryo cavity of F2 (intercross of B6D2F1) or CD-1 (albino) female miceat 3.5 d (days postcoitum). Host eight-cell embryos were collected fromfemale mice at 2.5 d, and 7-10 CiPS cells were injected into eachembryo. After injection, blastocysts and eight-cell embryos (6-8 embryosin each oviduct or horn of the uterus) were transferred into 2.5 d or0.5 d pseudopregnant CD-1 females, respectively. Chimeric mice wereidentified by coat color and then assessed for germline transmission bymating with ICR mice.

XEN Chimera Assay

GFP-labeled XEN-like cells were induced from the GFP-labeled MEFisolated from GFP (ICR×ICR) mice. For chimera test, GFP-labeled XEN-likecell colonies were picked and disaggregated to single cells by 0.25%trypsin-EDTA. Approximately 10 to 15 XEN-like cells were injected intoblastocysts and transferred to the uterus of E2.5 pseudopregnantfemales. Chimera conceptus between E6.5-8.5 were dissected carefully tokeep the parietal yolk sac intact and observed with fluorescencestereoscopy.

For the chimera test with eXENs and CeXENs, cells were infected withlentiviral vectors expressing EGFP and FASC sorted for the purificationof EGFP-positive cells.

DNA Microarray and RNA-Seq

Total mRNA was isolated from mouse fibroblasts, CiPSCs and ESCs.Microarrays were performed as reported by Li, et al., Cell Res.,21:196-204 (2011). RNA sequencing libraries were constructed using theIllumina mRNA-seq Prep Kit (Illumina). Fragmented and randomly primed200 bp paired-end libraries were sequenced using Illumina HiSeq 2000.Hierarchical clustering of the microarray data was performed as reportedby Li, et al., Cell Res., 21:196-204 (2011). Heatmaps were generatedusing R (Bioconductor). In some studies, total RNA was isolated usingthe RNeasy Plus Mini Kit (Qiagen). RNA sequencing libraries wereconstructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®(NEB). Fragmented and randomly primed 200 bp paired-end libraries weresequenced using Illumina HiSeq 2500. Hierarchical clustering, scatterplots and heatmaps were generated in R v3.2.0 using the amap package,the graphics package and the pheatmap package, respectively.

Bisulfite Genomic Sequencing

Genomic DNA was modified by bisulfate treatment and purified using theMethylCode™ Bisulfite Conversion Kit (Invitrogen) according to themanufacturer's protocol. The primers are listed in Table 2. Theamplified fragments were cloned into the pEASY-blunt Vector (Transgene).Ten randomly picked clones from each sample were sequenced.

cAMP, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH)Quantification

cAMP was quantified using the Direct cAMP ELISA Kit (Enzo) according tothe manufacturer's protocol. For SAM and SAH quantification, culturedcells (1,000,000 cells) were trypsinized and homogenized byultrasonication in 200 μl PBS. Then 40 μl of 400 mg/ml TCA was added.Cell extracts were incubated on ice for 30 min. After centrifugation at4° C. (13,000 rpm, 15 min), the supernatants were filtered through a0.22 μm filter and analyzed by high-performance liquid chromatography(HPLC, Shimadzu) with HILIC columns (Waters).

Comparative Genomic Hybridization (CGH) Analysis.

For CGH experiments, genomic DNA was extracted and hybridized toNimbleGen 3×720K mouse whole-genome tiling arrays by Imagenes usingC57BL/6 MEF DNA as a reference (Gene BioDesign).

Flow Cytometry Analysis

Cultured cells were trypsinized into single cells and then resuspendedin PBS containing 3% fetal bovine serum. Using endogenous Oct4-GFP, FACSanalyses were performed with a FACSCalibur instrument (BD Biosciences).The data were analyzed with FCS Express 4 (De Novo).

Chromatin Immunoprecipitation (ChIP)

ChIP was performed using the EZ-Magna ChIP A/G Kit (Millipore) accordingto the manufacturer's protocol. Anti-H3K27me3 (Abcam, ab6002),anti-H3K9me2 (Millipore, 07-441), anti-H3K4me3 (Abcam, ab8580) andanti-H3K9ac (Abcam, ab4441) antibodies were used. Followingimmunoprecipitation, DNA was analyzed by real-time PCR. The primers usedare listed in Table 2.

Southern Blot

Southern blot was performed with the DIG High Prime DNA Labeling andDetection Starter Kit II (Roche, 11 585 614 910), with reference to“Roche Techniques for Hybridization of DIG-labeled Probes to a Blot”. 20μg genomic DNA isolated from iPS cells or MEFs was digested with EcoRIand XbaI. The DNA probe was designed based on psi sequence, which ispresent in the pLL3.7-_U6 vector and Fu-tet vectors and could thus beintegrated into the genome along with exogenous transgenes after virusinfection.

Luciferase Activity Assays

MEFs were plated at a density of 40,000 cells per well of a 24-wellplate and transiently transfected with Oct4 promoter reporters usingLipofectamine LTX & Plus Reagent (Invitrogen) according to themanufacturer's instructions. pRL-TK plasmids (Promega) werecotransfected in each well as internal references, and the total DNAconcentrations for all transfections were equalized by adding emptypLL3.7-_U6 vector. At 48 hours after transfection, cells were washed inPBS and lysed in passive lysis buffer (Promega). Luciferase activity wasmeasured with the Dual-luciferase Reporter Assay System (Promega) usinga Centro LB960 96-well luminometer (Berthold Technologies) andnormalized to Renilla luciferase activity. Empty expression vectorplasmids were used as negative control. The fold activation describesthe ratio of firefly to Renilla luciferase activity for each conditioncompared with that of the empty vector control.

Western Blot Analysis

Cells were cultured in 100 mm dishes, washed in PBS and scraped in lysisbuffer. Aliquots were loaded onto an 8-10% SDS-polyacrylamide gel andblotted onto a nitrocellulose membrane. Membranes were incubatedovernight at 4° C. with rabbit anti-EZH2 (Abcam, ab3748) at a dilutionof 1:1000. Goat anti Rabbit IgG(H+L)/HRP (ZSBIO, ZB-2301) was used asthe secondary antibody. Detection was performed using SuperSignal WestPico solutions (Pierce).

Example 1: Chemical Substitutes for Oct 4

To identify chemical substitutes of Oct4, MEFs from OG mice were platedat a density of 20,000 cells per well of a 12-well plate and infectedwith lentiviruses encoding Sox2, Klf4 and c-Myc. After infection, themedium was replaced with LIF-free ESC culture medium. Individualchemicals from small-molecule libraries were added to each well. Themedium and chemicals were changed every 4 days. Chemical treatments werecontinued for 14-20 days or until GFP-positive colonies appeared.Primary hits were selected for further confirmation and optimization.

Small molecules that enable reprogramming in the absence of Oct4 weresearched using Oct4 promoter-driven green fluorescent protein (GFP)expression (OG) mouse embryonic fibroblasts (MEFs), with viralexpression of Sox2, Klf4, and c-Myc. After screening up to 10,000 smallmolecules (Table 1C), Forskolin (FSK), 2-methyl-5-hydroxytryptamine(2-Me-5HT), and D4476 (Table 1D) were identified as chemical“substitutes” for Oct4 (FIG. 1A to 1F).

Passaged SKM-FSK-iPSCs exhibit typical ESC morphology and homogeneouslyexpress GFP (FIG. 1B). The SKM-FSK-iPSCs can contribute to chimericmice, including gonadal tissues. Similarly, iPSCs induced by SKM with2-Me-5HT treatment can contribute to chimeric mice.

A small molecule combination “VC6 T” [VPA, CHIR99021 (CHIR), 616452,tranylcypromine], that enables reprogramming with a single gene, Oct4(Li, et al., Cell Res., 21:196-204 (2011)), was used next to treatOG-MEFs plus the chemical substitutes of Oct4 in the absence oftransgenes. The data shows that VC6 T plus FSK (VC6TF) induced someGFP-positive clusters expressing E-cadherin, a mesenchyme-to-epitheliumtransition marker, reminiscent of early reprogramming by transcriptionfactors (Li, et al., Cell Stem Cell, 7:51-63 (2010); Samavarchi-Tehrani,et al., Cell Stem Cell, 7:64-77 (2010)) (FIG. 2). However, theexpression of Oct4 and Nanog was not detectable, and their promotersremained hypermethylated, suggesting a repressed epigenetic state (FIGS.2A and 2B).

Example 2: Small Molecules that Facilitate Late Reprogramming

To identify small molecules that facilitate late reprogramming, adoxycycline (DOX)-inducible Oct4 expression screening system was used(Li, et al., Cell Res. 21:196-204 (2011)).

MEFs from OG mice were plated as described above and infected withFu-tet-hOct4 and FUdeltaGW-rtTA lentiviruses. The induction protocol wascarried out as described above.

After infection, the culture medium was replaced with LIF-free ESCculture medium containing VC6 T (VPA, CHIR99021, 616452,Tranylcypromine) plus DOX (1 μg/ml). Alternatively, MEFs harboringDOX-inducible Oct4 from Tet-On POU5F1 mouse strainB6;129-Gt(ROSA)26Sor^(tm1(rtTA*M2)Jae)Col1a₁ ^(tm2(tetO-Pou5f1)Jae)/Jwere used in this screen (Li, et al., Cell Res., 21:196-204 (2011)).These two DOX-inducible systems were only used in this screen, but notin complete chemical reprogramming. Individual chemicals fromsmall-molecule libraries were added to each well. The concentrations ofsmall molecules are listed in Table 1D. Small molecules were added atdifferent culture time points. 5-aza-C(5-Azacytidine) and DZNep wereadded from day 8. The medium and chemicals were changed every 4 days;DOX was added only for the first 4-8 days. Chemical treatments werecontinued for 16-24 days or until GFP-positive colonies appeared.Primary hits were selected for further confirmation and optimization.CiPSC colonies were counted on day 44. Primary hits were selected forfurther confirmation and optimization.

Small molecule hits, including several cAMP agonists (FSK, ProstaglandinE2, and Rolipram) and epigenetic modulators [3-deazaneplanocin A(DZNep), 5-Azacytidine, sodium butyrate, and RG108], were identified inthis screen (FIG. 3 and Table 1D).

Example 3: Complete Chemical Reprogramming without the Oct-4 InducibleSystem

To achieve complete chemical reprogramming without the Oct4-induciblesystem, small molecules were further tested in the chemicalreprogramming of OG-MEFs without transgenes. When DZNep was added 16days after treatment with VC6TF (VC6TFZ), GFP-positive cells were

obtained more frequently by a factor of up to 65 than those treated withVC6TF, forming compact, epithelioid, GFP-positive colonies withoutclearcut edges (FIGS. 4A-B and 4E). In these cells, the expressionlevels of most pluripotency marker genes were elevated but were stilllower than in ESCs, suggesting an incomplete reprogramming state (FIGS.4G and H). After switching to 2i medium with dual inhibition (2i) ofglycogen synthase kinase-3 and mitogen-activated protein kinasesignaling after day 28 post treatment, certain GFP-positive coloniesdeveloped an ESC-like morphology (domed, phase-bright, homogeneous withclear-cut edges) (FIG. 4C) (Silva, et al., PLoS Biol., 6:e253 (2008);Theunissen, et al., Curr. Biol., 21:65-71 (2011)). These colonies couldbe further cultured for more than 30 passages, maintaining an ESC-likemorphology (FIGS. 4A, 4D and 4G). These are referred to as 2i-competent,ESC-like, and GFP-positive cells as chemically induced pluripotent stemcells (CiPSCs). A schematic diagram for the formation of CiPSCs asdescribed above is shown in FIG. 4F.

Next, the dosages and treatment duration of the small molecules wereoptimized leading to generation of 1 to 20 CiPSC colonies from 50,000initially plated MEFs (FIGS. 5A-K). FIGS. 5A-F show the potentialconcentrations of VPA, CHIR99021, 616452, tranylcypromine and Forskolinin inducing CiPSCs. They also indicate the reprogramming efficiency maydiffer from experiments using different concentrations of the smallmolecules. The preferable concentrations for each small molecule wereshown (VPA, 0.5 mM; CHIR99021, 10 μM; 616452, 10 μM; Forskolin, 50 μMand DZNep 50 nM). FIG. 5G shows the preferable concentration of bFGF(100 ng/mL), it also indicate that bFGF is necessary in inducing CiPSCs.FIG. 5H, I, J show the preferable durations of the small molecules andbFGF that were used in inducing CiPSCs. FIG. 5K indicates the preferabletime points to change the medium containing VC6TF into 2i-medium. FIG. 5A-F show the potential concentrations of VPA, CHIR99021, 616452,tranylcypromine and Forskolin in inducing CiPSCs. They also indicate thereprogramming efficiency may differ from experiments using differentconcentrations of the small molecules. The preferable concentrasions foreach small molecule were shown (VPA, 0.5 mM; CHIR99021, 10 μM; 616452,10 μM; Forskolin, 50 μM and DZNep 50 nM). FIG. 5G shows the preferableconcentration of bFGF (100 ng/mL), it also indicate that bFGF isnecessary in inducing CiPSCs. FIG. 5H, I, J show the preferabledurations of the small molecules and bFGF that were used in inducingCiPSCs. FIG. 5K indicates the preferable time points to change themedium containing VC6TF into 2i-medium.

After an additional screen, some small-molecule boosters of chemicalreprogramming were identified, among which, a synthetic retinoic acidreceptor ligand, TTNPB, enhanced chemical reprogramming efficiency up toa factor of 40, to a frequency comparable to transcriptionfactor-induced reprogramming (up to 0.2%) (FIGS. 6A-C; and Table 1D). Anadult chimeric mouse produced with CiPSCs derived from MAFs (cloneMAF-CiPS-84) (F) and black F2 offsprings produced with CiPSCs derivedfrom MAFs (clone MAF-CiPS-85). Genomic PCR analysis showed that CiPSCswere free of transgene contamination (FIGS. 6D and E).

Using the small-molecule combination VC6TFZ, CiPSC lines were obtainedfrom mouse neonatal fibroblasts (MNFs), mouse adult fibroblasts (MAFs),and adipose-derived stem cells (ADSCs) with OG cassettes by efficiencylower by a factor of −10 than that obtained from MEFs. Table 5.

TABLE 5 Summary of CiPS cell characterization Gene Clone Initial cellChemical ESC-like and Non- AP expression Germ-line number types Mousestrain combinations GFP-positive transgenic staining RT-PCRimmunostaining profiling Teratoma karyotype DNA chimeras transmissionCiPS-6 MEFs OG (C57) × ICR VC6TFMB + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ — ✓ No CiPS-11MEFs OG (C57) × ICR VC6TFMB + Z ✓ ✓ ✓ ✓ ✓ ✓ — — ✓ — — CiPS-21 MEFs OG(C57) × ICR VC6TFDBS + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ CiPS-25 MEFs OG (C57) ×ICR VC6TFMDBSPR + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ — CiPS-30 MEFs OG (C57) × ICRVC6TFMDBSPR + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ — — CiPS-34 MEFs OG (C57) × ICRVC6TFMP + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ CiPS-36 MEFs OG (C57) × ICR VC6TFMDB +Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ No CiPS-39 MEFs OG (C57) × ICR VC6TFMDB + Z ✓ ✓ ✓✓ ✓ — — ✓ — ✓ — CiPS-42 MEFs OG (C57) × ICR FC6 + Z ✓ ✓ ✓ ✓ ✓ — ✓ — — —— CiPS-43 MEFs OG (C57) × ICR FC6 + Z ✓ ✓ ✓ ✓ ✓ — ✓ — — — — CiPS-44 MEFsOG (C57) × ICR FC6 + Z ✓ ✓ ✓ ✓ ✓ — — — — ✓ — CiPS-45 MEFs OG (C57) × ICRFC6 + Z (w/o bFGF) ✓ ✓ ✓ ✓ ✓ ✓ — ✓ ✓ ✓ — CiPS-47 MEFs OG (C57) × ICRVC6TF + Z ✓ ✓ ✓ ✓ ✓ — ✓ — ✓ ✓ — CiPS-50 MEFs OG (C57) × ICR VC6F + Z ✓ ✓✓ ✓ ✓ ✓ ✓ — ✓ ✓ — CiPS-56 MEFs OG (C57) × ICR VC6TFBPS + Z ✓ ✓ ✓ ✓ ✓ ✓ —— — — — CiPS-82 MEFs OG (C57) × OG VC6TF + Z ✓ ✓ ✓ ✓ ✓ — — — — ✓ — (C57)CiPS-453 MEFs OG (C57) × 129 VC6TFPS + Z ✓ ✓ ✓ ✓ — ✓ — — ✓ ✓ CiPS-WT1MEFs (without ICR VC6TFMPS + Z ✓ ✓ ✓ ✓ ✓ ✓ — — ✓ ✓ — OG-reporter)CIPS-WT2 MEFs (without C57 × 129 VC6TFMPS + Z ✓ ✓ ✓ ✓ ✓ ✓ — — ✓ ✓ —OG-reporter) MNF-CiPS- Mouse neonatal OG (C57) × ICR VCT6FMDBR + Z ✓ ✓ ✓✓ ✓ ✓ ✓ ✓ ✓ ✓ No 1 fibroblasts (MNFs) MNF-CiPS- MNFs OG (C57) × ICRVC6TFRP + Z ✓ ✓ ✓ ✓ ✓ — — — — ✓ — 2 MNF-CiPS- MNFs OG (C57) × ICRVC6TFS + Z ✓ ✓ ✓ ✓ ✓ ✓ — — ✓ ✓ No 7 ADSC- Adipocyte stem OG (C57) × ICRVC6TFMPS + Z ✓ ✓ ✓ ✓ ✓ — ✓ — — No — CiPS-1 cells ADSC- Adipocyte stem OG(C57) × ICR VC6TFMPS + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ — ✓ — — CiPS-2 cells ADSC-Adipocyte stem OG (C57) × ICR VC6TFDM + Z ✓ ✓ — ✓ — — ✓ — — ✓ — CiPS-3cells ADSC- Adipocyte stem OG (C57) × ICR VC6TFDM + Z ✓ ✓ — ✓ — — ✓ — ✓✓ — CiPS-4 cells MAF-CiPS- MAFs OG (C57) × ICR VC6TF + Z ✓ ✓ — ✓ ✓ — ✓ —✓ — — 1 MAF-CiPS- MAFs OG (C57) × ICR VC6TFDM + Z ✓ ✓ — ✓ ✓ ✓ ✓ — ✓ ✓ No3 MAF-CiPS- MAFs OG (C57) × ICR VC6TFBS + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ 62MAF-CiPS- MAFs OG (C57) × ICR VC6TFPS + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ 63MAF-CiPS- MAFs OG (C57) × ICR VC6TFBS + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ — ✓ ✓ — 73MAF-CiPS- MAFs OG (C57) × ICR VC6TFB + Z ✓ ✓ ✓ ✓ ✓ ✓ — — — ✓ — 76MAF-CiPS- MAFs OG (C57) × ICR FC6 + Z ✓ ✓ ✓ ✓ ✓ ✓ — — ✓ ✓ — 80 MAF-CiPS-MAFs OG (C57) × ICR FC6 + Z ✓ ✓ ✓ ✓ ✓ ✓ — — ✓ ✓ — 81 MAF-CiPS- MAFs OG(C57) × ICR VC6TFP + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ — 83 MAF-CiPS- MAFs OG (C57)× ICR VC6TFN + Z ✓ ✓ ✓ ✓ ✓ — ✓ ✓ ✓ ✓ — 84 MAF-CiPS- MAFs OG (C57) × ICRVC6TFN + Z ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ 85 CiPS-101 MEFs OG (C57) × ICR FC6 ✓ —— — — — — — — — — CiPS-102 MEFs OG (C57) × ICR FCT6 + Z ✓ — — — — — — —— — — CiPS-103 MEFs OG (C57) × ICR FC6N + Z ✓ — — — — — — — — — —CiPS-104 MEFs OG (C57) × ICR FC6T + 4PB + Z ✓ — — — — — — — — — —CiPS-105 MEFs OG (C57) × ICR VCT6 + Z ✓ — — — — — — — — — — CiPS-31 MEFsOG (C57) × ICR VC6TF ✓ ✓ ✓ ✓ ✓ — — — — — — CiPS-106 MEFs OG (C57) × ICRVC6T + DBcAMP + Z ✓ — — — — — — — — — — CiPS-107 MEFs OG (C57) × ICRVC6T + IBMX + Z ✓ — — — — — — — — — — CiPS-108 MEFs OG (C57) × ICRVC6T + Rloipram + Z ✓ — — — — — — — — — — CiPS-109 MEFs OG (C57) × ICRVF6T + TD114-2 + Z ✓ — — — — — — — — — — CiPS-110 MEFs OG (C57) × ICRVC6TF + NepA ✓ — — — — — — — — — — CiPS-111 MEFs OG (C57) × ICR VC6TF +Adox ✓ — — — — — — — — — — CiPS-112 MEFs OG (C57) × ICR VC6TF + DZA ✓ —— — — — — — — — — CiPS-113 MEFs OG (C57) × ICR VC6TF + Decitabine + ✓ —— — — — — — — — — EPZ004777 CiPS-114 MEFs OG (C57) × ICR VC6TFN + Z ✓ —— — — — — — — — — CiPS-115 MEFs OG (C57) × ICR VC6TF + AM580 + Z ✓ — — —— — — — — — — CiPS-116 MEFs OG (C57) × ICR VC6TF + Ch55 + Z ✓ — — — — —— — — — — CiPS-117 MEFs OG (C57) × ICR VC6TF + TTNPB + ✓ — — — — — — — —— — PGE2 + 5-aza-C CiPS-118 MEFs OG (C57) × ICR VC6TF + TTNPB + ✓ — — —— — — — — — — PGE2 + Decitabine CiPS-119 MEFs OG (C57) × ICR VC6TFDMB +UNC0638 + ✓ ✓ — ✓ ✓ — ✓ — — — — Scriptaid CiPS-120 MEFs OG (C57) × ICRVC6TF + Decitabine + ✓ — — — — — — — — — — EPZMoreover, CiPSCs were induced from wild-type MEFs without OG cassettesor any other genetic modifications by a comparable efficiency to thatachieved from MEFs with OG cassettes. The CiPSCs were also confirmed tobe viral-vector free by genomic polymerase chain reaction (PCR) andSouthern blot analysis (FIGS. 7A-D).

Furthermore, small molecule combinations were used to generate CiPSCsfrom neural stem cells and from cells obtained from the intestinalepithelium (Table 6).

TABLE 6 Generation of CiPSCs from neural stem cells and cells from theintestinal epithelium Clone Initial cell ES-like genomic AP number typemouse strain small molecules morphology PCR staining RT-PCR IE-CiPS-intestinal OG (C57) × ICR VC6TFAM580 + ✓ ✓ ND ✓ 1 epithelium Z cellNS-CiPS- neural stem OG (C57) × ICR VC6TFZ + Ch55 + ✓ ✓ ND ✓ 1 cellDecitabine + EPZ Clone DNA chimeric germ-line number immunofluorescencemicroarray teratoma karyotype methylation mice transmission IE-CiPS- ✓ ✓✓ ✓ ✓ ✓ ✓ 1 NS-CiPS- ✓ ND ✓ ND ND ✓ ND 1 (“ND” represents “notdetermined yet”)

AM580 is4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoicacid]; Ch55 is4-[(1E)-3-[3,5-bis(1,1-Dimethylethyl)phenyl]-3-oxo-1-propenyl]benzoicacid] and EPZ. Intestinal and neural stem cells, likes MEF can bereprogrammed using VC6TFZ, without addition of the indicated smallmolecules, albeit with a different efficiency.

Experiments were next carried out to determine which of these smallmolecules were critical in inducing CiPSCs. Four essential smallmolecules (shown below) whose individual withdrawal from thecombinations generated significantly reduced GFP-positive colonies andno CiPSCs were identified (FIGS. 8A-B).

These small molecules (C6FZ) are: CHIR (C), a glycogen synthase kinase 3inhibitor (Ying, et al., Nature, 453:519-523 (2008)); 616452 (Zhang, etal., Cell Res. 21:196-204 (2011)), a transforming growth factor-betareceptor inhibitor (Maherali, et al., Curr. Biol., 19:1718-1723 (2009));FSK (F), a cAMP agonist (FIG. 14F) (Insel, et al., Cell. Mol.Neurobiol., 23:305-314 (2003)); and DZNep (Z), an S-adenosylhomocysteine(SAH) hydrolase inhibitor (FIG. 14A) (Chiang, et al., Pharmacol., Ther.77:115-134 (1998); Gordon, et al., Eur. J. Biochem., 270:3507-3517(2003)).

C6FZ was able to induce CiPSCs from both MEFs and MAFs, albeit by anefficiency lower by a factor of 10 than that induced by VC6TFZ (Table5).

The effect of inhibiting adenylate cyclase on the number of coloniesgenerated by VC6TFZ was tested. Inhibition of adenylate cyclase by2'S′ddAdo decreased the number of colonies formed by VC6TFZ (FIG. 14F).The effect of replacing FSK with the cAMP analog (DBcAMP), alone or incombination with phosphodiesterase inhibitors, IBMX, or thephosphodiesterase inhibitor, rolipram and IBMX, on cell reprogrammingwas also tested. Both DBcAMP and IBMX were used at 50 μM in thecombination treatment. The data is shown in FIGS. 14G-J. Theintracellular levels of cAMP concentrations in MEF following treatmentwith rolipram (10 uM), IBMX (50 μM, DBcAMP (50 μM) and FSK (10 μM) wascompared to control (−) DMSO (FIG. 14K). Forskolin significantlyelevates the cAMP level, which is similar to other cAMP agonists(Rolipram and IBMX) or analog (DBcAMP). GFP+ Pluripotent stem cells wereinduced with other cAMP agonists or analog, which were used tosubstitute Forskolin. A cAMP inhibitor, 2'S′ddAdo suppressed theefficiency of CiPSC induction. These taken together suggest thatForskolin facilitate chemical reprogramming by modulating cAMP signalingpathway.

Generation of CiPSCs from NSCs Obtained from Oct4-GFP Transgenic Mice

In other experiments NSCs (obtained from Oct4-GFP transgenic mice) weretreated with the reprogramming cocktail “VC6TF” (VPA, V; CHIR99021,CHIR, C; 616452, 6; Tranylcypromine, T; Forskolin, F). This did not ableto induce pluripotency from NSCs. Then, potential reprogramming boostersincluding a RA agonist, Ch 55 (5) and a Dotl1 inhibitor, EPZ 004777(EPZ, E) were added i.e., VC6TFZE5. The number of CiPSC coloniesobtained from 30,000 with different RAR agonists is shown in FIG. 14N.Epithelial clusters emerged on day 8 after adjusting the concentrationof 616452 from the traditional 10 μM to 2 μM (data not shown), andproliferated with time. After the addition of DZNep (Z) from day 20,compact and epithelioid colonies were observed on day 32 (data notshown). Subsequently, the Oct4-GFP reporter was gradually turned onafter switching these colonies into 2i-medium with dual inhibition (2i)of MAPK signaling and GSK3 from day 40, and ESC-like OG-positivecolonies with clear-cut edges were observed. The final cells arereferred to as NSC-CiPSCs. The same small molecule combination was usedto obtain CiPSCs from NSCs of postnatal mice (data not shown). PrimaryNSC-CiPSC colonies were harvested and passaged in 2i-medium plus mouseleukemia inhibitory factor (LIF) for more than 20 passages with stableESC-like morphology and Oct4-GFP expression. There studies indicatedthat selection of the concentration of 616452 between the early and latestages of reprogramming affects the number of CiPSC obtained withchemical reprogramming from NSCs (14 L). At the late stage, theconcentration of 616452 should be readjusted to 5 μM from day 20 (datanot shown).

Generation of CiPSCs from Small Intestinal Epithelial Cells Obtainedfrom Oct4-GFP Transgenic Mice

IECs isolated from the small intestinal tissue of Oct4-GFP transgenicmice at embryonic day 13.5 were used in these studies. The isolated IECsexhibited epithelial cell morphology, and immunofluorescence stainingshowed that the IECs highly expressed a specific intestinal epithelialcell marker, KERATIN 20 (KRT20) (data not shown).

Isolated IECs were cultured with the reported chemical reprogrammingcocktail VC6TF and the RAR agonist AM 580. DZnep was then added to thecocktail from day 16-20. During this process, epithelioid clusters wereobserved from day 4-8, and formed colonies from day 16 (data not shown).After switching to 2i-medium from day 32-36, compact, epithelioid,ESC-like OG-positive colonies with clear-cut edges were developed (datanot shown). Primary CiPSC colonies were calculated and harvested on day44-46, and passaged in 2i-medium plus mouse LIF for more than 20passages, maintaining ESC-like morphology (data not shown). These cellswere referred to as IEC-CiPSCs.

A lineage tracing experiment was performed using transgenic miceexpressing the Cre recombinase driven by Villin, an epithelium specificgene promoter, crossed with mice expressing a loxP-stop-loxP-td-Tomatolocated in Rosa26 locus. The IECs were labelled by tdTomato fluorescence(data not shown). After exposure to the chemical cocktail, tdTomatofluorescent CiPSC colonies were generated from IECs (FIG. 14M and datanot shown). By contrast to the data shown NSC, increasing theconcentration of 616452 up to 20 μM during the first 12 days bestsupport IEC reprogramming, whereas a decreased concentration of 616452is beneficial for NSC reprogramming (FIG. 14L bottom panel).

Example 4: Characterization of CiPSC Lines

Characterization of CiPSC Obtained from Fibroblasts

The established CiPSC lines were then further characterized. They grewwith a doubling time (14.1 to 15.1 hours) similar to that of ESCs (14.7hours), maintained alkaline phosphatase activity, and expressedpluripotency markers, as detected by immunofluorescence and reversetranscription (RT)-PCR (FIG. 9A-9D). Specifically, CiPS-25 from passage21, CiPS-26 from passage 7 and CiPS-30 from passage 22 were cultured foranother 6 passages. ESCs (R1) from passage 29 were used as controls.Cells were passaged every three days and seeded at a density of 20,000cells per well in a 12-well plate without feeder layers in 2i-medium.Error bars indicate the s.d. (n=3). The calculated population doublingtimes of these cells were 14.8±2.1 (CiPS-25), 15.1±1.9 (CiPS-26), and14.1±1.7 (CiPS-30) hours. These times were equivalent to that of ESCs(14.7±1.2 hours).

The gene expression profiles were similar in CiPSCs, ESCs, andOSKM-iPSCs (iPSCs induced by Oct4, Sox2, Klf4, and c-Myc). DNAmethylation state and histone modifications at Oct4 and Nanog promotersin CiPSCs were similar to that in ESCs. In addition, CiPSCs maintained anormal karyotype and genetic integrity for up to 13 passages, i.e., CiPScells maintain normal chromosome numbers, few copy number variations andgenetic mutations, making them safe for further clinical application.

To characterize their differentiation potential, CiPSCs were injectedinto immunodeficient (SCID) mice. The cells were able to differentiateinto tissues of all three germ layers-respiration epithelium (endoderm);muscle cells (mesoderm); neural epithelium (endoderm) and pigmentedepithelium (ectoderm). When injected into eight-cell embryos orblastocysts, CiPSCs were capable of integration into organs of all threegerm layers, including gonads and transmission to subsequentgenerations. An adult chimeric mouse was produced from clone ciPS-34 aswell as F2 offspring. Germline contribution of clone CiPS-45 was shownin testes. An adult chimeric mouse was also produced with CiPSCs derivedfrom MNFs (clone MNF-CiPS-1). An adult chimeric mouse was produced withCiPSCs derived from MAFs (clone MAF-CiPS-62). Black F2 offspring wereproduced with CiPSCs derived from MAFs (clone MAF-CiPS-62). Black F2offsprings were produced with CiPSCs derived from MAFs (cloneMAF-CiPS-63). Chimeras were produced with CiPSCs derived from WT MEFs(clone CiPS-WT1, ICR) that were microinjected into (C57×DBA)×ICRembryos. Unlike chimeric mice generated from iPSCs induced bytranscription factors including c-Myc (Nakagawa, et al., Proc. Natl.Acad. Sci. U.S.A. 107:14152-14157 (2010)), the chimeric mice generatedfrom CiPSCs were 100% viable and apparently healthy for up to 6 months(FIG. 10A). These observations show that the CiPSCs were fullyreprogrammed into pluripotency (Table 5).

Characterization of NSC- and IEC-Derived CiPSCs

The established NSC-CiPSC lines and IEC-CiPSC lines were furthercharacterized. The doubling time of the established CiPSC lines was 18h-24 h, similar to mouse ESCs (FIG. 14M). Pluripotency gene expressionsin derived CiPSC lines were comparable to mESCs as detected byquantitative real-time PCR and immunofluorescence staining, p (FIG.10B-C). Global gene expression analysis was performed by RNA-sequencing,and NSC-CiPSCs and IEC-CiPSCs clustered with ESCs, in contrast toinitial cells (data not shown). To detect the epigenetic reprogrammingstatus, DNA methylation analysis was performed at two core pluripotencygenes, the Oct4 and Nanog promoters, by bisulfate sequencing. The resultshowed that Oct4 and Nanog promoters were hypomethylated in CiPSCs andmESCs (data not shown). In addition, normal karyotypes were maintainedfor up to 7 passages (data not shown), and Table 7.

TABLE 7 Number of chromosomes in NSC-CiPSCs and IEC-CiPSCs by karyotypeanalysis Chromosome number Sample 2n = 40 2n < 40 2n > 40 NSC-CiPSC ♀100% 0% 0% IEC-CiPSC 

 98% 2% 0% pVillin-Cre-tdTomato 100% 0% 0% IEC-CiPSC 

To evaluate the pluripotency of CiPSCs derived from NSCs and IECs, theirin vivo developmental potential was examined. Both CiPSC lines couldform well-differentiated teratomas with tissues from all three germlayers after injection into immunodeficient (NOD/SCID) mice (data notshown). When injected into blastocysts, CiPSCs were able to generatechimeric mice with germline transmission competency (data not shown).These results demonstrated that CiPSC lines derived from NSCs and IECswere pluripotent and fully reprogrammed.

Example 5: Pluripotency Inducing Properties of Small Molecules

To better understand the pluripotency-inducing properties of these smallmolecules, the global gene expression during chemical reprogramming wasprofiled. To determine clustering of gene expression profiles duringchemical reprogramming, cell culture samples treated with VC6TFZ (Z wasadded from day 20) during chemical reprogramming on day 12, 20 and 32were analyzed. MEFs on day 0, CiPSCs and ESCs were used as controls.Sequential activation of certain key pluripotency genes was observed,which was validated by real-time PCR and immunofluorescence. Genes thatexpress in ESCs by more than 10 fold and in samples (day 32) by morethan 3 fold compared to MEFs (day 0) include Sall4, Sox2, Lin28a, Dppa2,Esrrb, Klf4 and Pou5f1. Genes that express in samples (day 32) by morethan 3 folds compared to MEFs (day 0) and ESCs include Sox 17, Gata6 andGata4. The expression levels of two pluripotency-related genes, Sall4and Sox2, were most significantly induced in the early phase in responseto VC6TF, as was the expression of several extra-embryonic endoderm(XEN) markers Gata4, Gata6, and Sox17 (FIG. 11A-J). The expression ofSall4 was enhanced most significantly as early as 12 hours after smallmolecule treatment, suggesting that Sall4 may be involved in the firststep toward pluripotency in chemical reprogramming (FIG. 11D). Withrespect to experiments in which small molecules were withdrawn from thetreatment used, the data shows that each of Forskolin, CHIR99021 and616452 is essential in activating the endogenous expression of Sall4 andSox2 after 12 days (FIGS. 11G and 11H), and the subsequent expression ofother pluripotency genes (FIG. 11I). These molecules were also requiredin activating XEN-genes, such as Gata6, Gata4 and Sox17.

The roles of the endogenous expression of these genes in chemicalreprogramming were examined, using gene overexpression and knockdownstrategies. The data shows that the concomitant overexpression of Sall4and Sox2 was able to activate an Oct4 promoter-driven luciferasereporter (FIGS. 12A-C) and was sufficient to replace C6F in inducingOct4 expression and generating iPSCs (FIGS. 12D-E). The endogenousexpression of Sall4, but not Sox2, requires the activation of the XENgenes, and vice versa (FIGS. 13A-D). This suggests a positive feedbacknetwork formed by Sall4, Gata4, Gata6, and Sox17, similar to thatpreviously described in mouse XEN formation (Lim, et al., Cell StemCell, 3:543-554 (2008)). Knockdown of Sall4 or these XEN genes impairedOct4 activation and the subsequent establishment of pluripotency (FIGS.13E-F), inconsistent with previous finding that Gata4 and Gata6 cancontribute to inducing pluripotency (Shu, et al., Cell, 153:963-975(2013)). Taken together, these findings revealed a Sall4-mediatedmolecular pathway that acts in the early phase of chemical reprogramming(FIG. 13G). This step resembles a Sall4-mediated dedifferentiationprocess in vivo during amphibian limb regeneration (Neff, at al., Dev.Dyn., 240:979-989 (2011)).

Next, the role of DZNep, which was added in the late phase of chemicalreprogramming was investigated. The data shows that Oct4 expression wasenhanced significantly after the addition of DZNep in chemicalreprogramming (FIG. 11A), and DZNep was critical for stimulating theexpression of Oct4 but not the other pluripotency genes (FIG. 11K). Asan SAH hydrolase inhibitor, DZNep elevates the concentration ratio ofSAH to S-adenosylmethionine (SAM) and may thereby repress theSAM-dependent cellular methylation process (FIG. 14A). Replacement ofDZNep by SAH hydrolase inhibitors ((−) Neplanocin A (Nep A), Adenozineperiodate (oxidized) Adox and 3-deazaadenosine (DZA)) ((Chiang, et al.,Pharmacol. Ther., 77:115-134 (1998), Gordon, et al., Eur. J. Biochem.270:3507-3517 (2003)), in combination with VC6TF treatment to induceCiPSC generation is shown in FIG. 14B The data shows that NEPA, ADOX andDZA are each useful in replacing DZNep in reprogramming. They modulatethe same target as DZNep, and can substitute DZNep in generating CiPSCs.

Consistently, DZNep significantly decreased DNA methylation and H3K9methylation (FIG. 11L) at the Oct4 promoter, which may account for itsrole in Oct4 activation (Feldman, et al., Nat. Cell Biol., 8:188-194(2006); Chen, at al., Nat. Genet., 45:34-42 (2013)). The function ofDZNep in inducing CiPSCs could not be replaced by down-regulating Ezh2expression (FIGS. 14C-E). GFP+/ES-like colonies in the primary culture,unlike other colonies, express high mRNA level of Nanog and low level ofGata6, resembling ESCs and the established CiPSCs (data not shown). Asmaster pluripotency genes, Oct4 and Sox2 may thereby activate otherpluripotency-related genes and fulfill the chemical reprogrammingprocess, along with the activation of Nanog and the silencing of Gata6,in the presence of 2i (Silva, et al., PLoS Biol. 6:e253 (2008),Theunissen, et al., Curr. Biol., 21:65-71 (2011). Boyer, et al., Cell,122:947-956(2005); Chazaud, at al., Dev. Cell, 10:615-624 (2006)).

In summary, as a master switch governing pluripotency, Oct4 expression,which is kept repressed in somatic cells by multiple epigeneticmodifications, is unlocked in chemical reprogramming by the epigeneticmodulator DZNep and stimulated by C6F-induced expression of Sox2 andSall4 (FIG. 13G).

Initial Gene Activation was Conserved in Chemical Reprogramming fromDifferent Cell Types

At the initial stage of chemical-induced reprogramming to pluripotency,NSCs and IECs were transformed into highly refractive phase-bright andepithelial-like cells, which share similar morphology of partialcolonies during MEF chemical reprogramming. Compact epithelioid colonieswere observed around day 20-32 (data not shown), and all CiPSC colonieswere derived from these epithelioid colonies in at least ten independentexperiments. Time-course quantitative real-time PCR was performed duringthe chemical reprogramming of NSCs and IECs. The data analysis resultsshowed that during reprogramming of NSCs and IECs, a pluripotency geneSall4, and differentiation-associated genes Gata4, Gata6 and Sox17, wereactivated as early as day 4 and increased with time in the early stage,while other pluripotency genes such as Lin28a, Dppa2, Esrrb and Oct4were activated much later (FIGS. 15B, 15C and 15E), which is similar tothe chemical reprogramming process from MEFs. Additionally, no innateexpression of Sall4, Gata4, Gata6 and Sox17 were detected in NSCs FIG.15D and data not shown). Interestingly, fine-tuning the concentration of616452, which is critical for the reprogramming of different cell typesreprogramming, results in higher gene expression of Sall4, Gata4 andSox17 (FIGS. 15F and 15D). These results suggest that the chemicalreprogramming from cell types of all three germ layers shares thesimilar initial gene activation programs, regardless of cell origins andinnate cellular features.

This proof-of-principle study demonstrates that somatic reprogrammingtoward pluripotency can be manipulated using only small-moleculecompounds (FIG. 15A). It established that the endogenous pluripotencyprogram can be established by the modulation of molecular pathwaysnonspecific to pluripotency via small molecules rather than byexogenously provided “master genes.” These findings understanding of theestablishment of cell identities and open up the possibility ofgenerating functionally desirable cell types in regenerative medicine bycell fate reprogramming using specific chemicals or drugs, instead ofgenetic manipulation and difficult-to-manufacture biologics.

In sum, the present data establishes that although a similar chemicalcocktail is generally required for different cell types, fine-tuning thetreatment of small molecules allows for improved reprogramminginitiation in different cell types. Especially for the chemicalreprogramming of NSCs, the concentration 616452, should be reduced to 2μM in comparison of 10 μM, which is used on MEFs. Notably, the reduced616452 concentration resulted in enhanced expression of Sall4 and Gata4genes (FIG. 15D), which further supports the role high expression ofthese genes in chemical initiation of pluripotency. In addition, 616452shows different function in chemical reprogramming and traditionaltranscription factor-induced reprogramming. In traditional transcriptionfactor-induced reprogramming, ectopic expression of Oct4 and Klf4 orc-Myc is sufficient to generate iPS cells from NSCs, in the absence ofexogenous Sox2 (Kim et al., 2008). Besides, 616452, also named asRepSox, was reported to substitute for Sox2 overexpression intraditional transcription factor-induced reprogramming (Ichida al.,2009). However, by contrast, in chemical reprogramming from NSCs, 616452cannot be removed, despite the high endogenous expression of Sox2. Inchemical reprogramming, 616452 rather facilitates the early expressionof Sall4, Gata4, Gata6 and Sox/7 and the subsequent epithelioid colonyformation (FIGS. 15F and 15D). These findings indicate that chemicalreprogramming is a different process from transcription factor-inducedreprogramming.

Interestingly, the reprogramming kinetics and frequency of NSCs and IECsare distinct from each other. The former underwent a longer early stage,with less epithelioid colonies generated, but almost 100 percent of thecolonies could be converted into CiPSC colonies. In contrast, the lattercould easily form more epithelioid colonies, but only 20-30 percent ofthese colonies were converted into CiPSC colonies.

Example 6. Identification of the Induction of Cell Colonies ExpressingXEN Cell Markers as a Cornerstone Event During Chemical Reprogramming

In the method for inducing pluripotent stem cells from non-pluripotentstem cells described in Hou et al., Science 341, 651-654 (2013)), thereare three essential stages in the chemical reprogramming process.Reprogramming of cells into CiPSC was obtained a cocktail of five smallmolecules, “VC6TF” was used in stage 1 for 16-20 days, following which,another small molecule, DZNep, was added at the start of stage 2 (i.e.,VC6TFZ) for the next 20-24 days, and 2i-medium was used in stage 3 forthe last 12-16 days, in some cases adding additional small molecules. Intotal, the chemical reprogramming process can take as long as 48-60days, resulting in a maximum of about 40 colonies (FIG. 19A).

In order to improve on the efficiency of reprogramming non-pluripotentcells into CiPSC, studies were conducted to characterize the chemicalreprogramming process, by carefully following the change in cellmorphology during chemical reprogramming in each stage. These studiesrevealed a number of epithelial colonies formed at the end of stage 1,which rapidly expanded during stage 2. By tracing the dynamic changes incell fate during chemical reprogramming, these studies revealed thatCiPSCs predominantly emerged from the inside of these epithelial cellcolonies (FIG. 16A). In some experiments, 100% of the CiPSCs weregenerated from these colonies, even when the cells were re-plated at alower density; the epithelial cell colonies had grown to less than 20%confluence. These findings indicate that there exist a subpopulation ofcells during reprogramming, which are better primed for conversion intoCiPSCs.

Immunofluorescence and quantitative real-time PCR (qRT-PCR) was thenused to examine the gene expression pattern of these epithelial cellcolonies. Immunofluorescence showed that all epithelial colonies formedin the end of stage 1 co-expressed SALL4, GATA4 and SOX17, master genesof XENs (Lim et al., Cell Stem Cell., 3:543-554 (2008)) (data notshown). qRT-PCR analysis further detected the expression of other XENmarker genes, such as Sox7 and Gata6 in these colonies (FIG. 16B), whichwere comparable to that of embryo-derived XEN cells (eXENs) (Kunath etal., 2005) (FIG. 16C). These epithelial cells expressing XEN markers arereferred to herein as XEN-like cells.

Studies were further conducted to determine whether these XEN-like cellsrepresent an intermediate state of chemical reprogramming. Using aXEN-expressing surface protein, EpCAM, XEN-like cells were enriched atday 20 by FACS sorting and found that selection for EpCAM-positive cellsgreatly enriched the proportions of cells forming XEN-like cell coloniesand subsequently generating CiPSCs by more than 20-fold (FIGS. 16C and16D). Similar results were obtained when the starting cells were neuralstem cells and intestinal epithelium cells. EpCAM also strongly enrichedthe cells that formed XEN-like colonies during chemical reprogrammingand improved the ability to generate CiPSCs from these two initial celltypes (FIG. 16E). To determine whether there exist transitional colonieswhich co-expressed XEN master genes and pluripotency-associated genes ifpluripotent stem cells were induced from XEN-like cells, expression oftwo markers was assessed in stage 2. Cells that co-expressed GATA4 andOCT4 were detected during stage 2 of reprogramming (data not shown).Cell colonies expressing GATA4 in the peripheral, and expressingpOct4-GFP in the middle of the colonies were identified in in stage 3,which could be the intermediate cell colonies of the cell fatetransition from XEN-like to pluripotent stem cells (data not shown).Together, these results indicate that the XEN-like cells represent anintermediate state of chemical reprogramming toward pluripotency.

Example 7. Identification of Small Molecules that Promote the Transitionfrom Fibroblasts to XEN-Like Cells

The identification of an intermediate state of chemical reprogramming inExample 6 provides a target for enhancing reprogramming conditions andfor screening novel small-molecule boosters in early reprogramming byusing XEN-like colony numbers as the readout. Increased concentration ofCHIR99021 is proved beneficial for the formation of XEN-like coloniesfrom MEFs (FIG. 17A). Through qRT-PCR analysis showed that thisincreased concentration of CHIR99021 promotes an up to 10-fold increasein the expression of XEN master genes Gata4 and Sox17 and an epitheliumcell marker, EpCAM (FIGS. 17B and 17C).

Next, the effects of a selected small-molecule library of previouslyreported reprogramming boosters was tested in the presence of asmall-molecule cocktail, VC6TF, with 20 μM CHIR99021, on thereprogramming of cell fate from fibroblasts to XEN-like cells. Among thetested small molecules, an RA agonist, AM580 (A), and a DOT1L inhibitor,EPZ004777 (E), each enhanced the formation of XEN-like colonies by 2 to3-fold When these small molecules were used together in a cocktail ofseven small molecules, VC6TFAE, the number of XEN-like colonies wasenhanced by more than 5-fold (FIG. 17D). These findings were furthervalidated by counting the numbers of SALL4 and GATA4 double-positivecolonies and by detecting the expression of XEN marker genes by qRT-PCR(FIG. 17E-I). Together, the numbers of XEN-like colonies, an indicatorof early reprogramming, could be enhanced more than 50-fold by selectinga combination of small molecule concentrations and additional smallmolecules type included in the cell culture medium.

Example 8. The Identification of Small Molecules that Promote theTransition from a XEN-Like to a Pluripotent State

Studies were next conducted to identify small molecules that facilitatethe transition of XEN-like colonies to CiPSCs during stages 2 and 3.Small-molecule screenings were performed in the presence of smallmolecule cocktail VC6TFA plus DZNep (Z) for 12 days with successfulenhanced generation of CiPSC colonies. By contrast, in Hou et al.,Science 341:651-654 (2013), the optimal duration for stage 2 was 20-24days; few CiPSC colonies were obtained if stage 2 was shortened to 12days.

After selecting and screening 88 small molecules, it was discovered thatCiPSC colonies formed in stage 3 only when the cell culture medium wassupplemented with 5-aza-dC during stage 2. 5-aza-dC (D) and EPZ004777(E) had synergistic effects and promoted the kinetics of stage 2. Thus,by using a cocktail of eight small molecules, VC6TFA+ZDE, for 12 daysduring stage 2, up to 20 CiPSC colonies were obtained from 100,000re-plated cells at the end of the reprogramming process of 44 days (FIG.18A). In contrast, few CiPSC colonies were obtained during the same timecourse without 5-aza-dC and EPZ004777. When another DOT1L inhibitor,SGC0946 (S) was used in place of EPZ004777 during stage 2, however, thereprogramming efficiency was increased further by as much as 5-fold at areplating density of 100,000 or 20,000 cells per well in a 6-well plate(FIG. 18A-C) (original cell density of 300,000 to 500,000 cells per wellin a 6-well plate), particularly when a further supplemented 2i-medium(N2B27-2i medium) was used (FIGS. 18B and 18C). Next, the effect if any,of replating density and concentration of small molecules the on thenumber of CiPSC colonies obtained was investigated. Using thesmall-molecule cocktail VC6TFA+ZDS during stage 2 for 12 days,approximately 100-600 CiPSC colonies was obtained from 50,000 re-platedcells during the final stage of chemical reprogramming. The data showedthat although SGC0946 can be more effective than EPZ004777 during stage2, SGC0946 could not substitute for EPZ004777 during stage 1 becausecell viability was decreased if SGC0946 was used from the start ofchemical reprogramming. (FIG. 18D-18G). Importantly, CiPSC coloniesemerged from 24-53% of the XEN-like colonies in five independentexperiments, indicating the transition ratio of a single XEN-like cellin the start of stage 2 to CiPSCs in the end of stage 3 (FIG. 18H-I). Inaddition, almost all CiPSC colonies were derived from XEN-like colonies,even though the efficiency was greatly improved (FIG. 18I).

These studies showed that the duration of the small-molecule treatmentand the re-plating cell density were both highly critical during thelater stages of reprogramming. The previous studies which did not followa reprogramming route of biasing/enriching the XEN-like cell populationfavors a re-plating cell density of 300,000 cells per well (Hou et al.,(2013)). However, when CiPSC reprogramming progresses via enriching forthe XEN-like cells, it is preferable to replate cells at a cell densityof 50,000-100,000 cells per well of a 6-well plate (FIG. 18D).Furthermore, the previously described protocol required 24 days ofreprogramming during stage 2 to achieve optimal reprogramming efficiency((Hou et al., (2013)). By contrast, a protocol which biases/enriches theXEN-like cell population as described herein requires an optimal stage 2duration of 12 days (FIG. 18E).

In summary, the methods disclosed herein greatly improve the celltransition from non-pluripotent into pluripotent cells, by selecting afirst cocktail of small molecules to enrich/bias cells to bereprogrammed towards the XEN-like state, selecting a second cocktail ofsmall molecules and replating density and cell culture time fortransition from XEN to CiPSCs.

Example 9. Establishment of a Robust CiPSC Induction Protocol wasEstablished Through Modulation of the Cell Transitions Through aXEN-Like State

Next, reprogramming conditions for stages 1, 2 and 3 identified inExample 8 were combined to reprogram fibroblasts into CiPSCs. Using thisnew protocol, a well of 50,000 initial fibroblasts was induced, and thecells were expanded to more than 1,000,000 or more re-plated cells(re-plated into 10-15 wells). A total of 1,000-9,000 CiPSC colonies wereobtained at the end of the reprogramming period with a total inductiontime of 40 days (16, 12, and 12 days for stages 1, 2 and 3,respectively). A comparison of the reprogramming protocol which includesbiasing towards XEN-like cells and the previous protocol exemplified inExample 3 is shown in FIG. 19A. Moreover, this new protocol wasreproduced independently more than 20 times, and CiPSCs could also begenerated from neonatal dermal fibroblasts (MNFs) and adult lungfibroblasts (MAFs) at a significantly enhanced efficiency (FIG. 19B).

The minimal time course required in inducing CiPSCs was further examinedby using this new small-molecule cocktail and the reprogrammingconditions established in Example 8. A minimum of 12 days were requiredin the formation of XEN-like colonies (cells were re-plated at day 8),and that at least another 14 days were required to induce CiPSCs fromXEN-like cells (FIG. 19C). In total, at the cost of efficiency, aminimum of 26 days are required to induce CiPSCs by using the newprotocol (FIG. 19C). In comparison, at least 44 days were required togenerate only 0-1 CiPSC colony from 40,000 initial cells using theoriginal protocol, and up to about 40 CiPSC colonies could be generatedif the treatment time frame was extended to more than 60 days (Table1A).

CiPSC colonies were then picked to establish CiPSC lines for furthercharacterization. As shown by immunostaining and qRT-PCR, the CiPSCsexpressed all the tested marker genes for pluripotent stem cells, suchas Oct4, Sox2 and Nanog (FIG. 19D, 19E). RNA-seq analysis showed thatCiPSCs induced with this protocol had gene expression profiles similarto those of ESCs (data not shown). CiPSCs were further tested for theirpotential for in vivo development. All tested 6 CiPSC lines were able toform teratoma after injection into SCID mice (data not shown) andgenerate chimeric mice after blastocyst injection (data not shown).Among 5 tested CiPSC lines, 4 lines showed germ line integrationpotential in chimeric mice (data not shown). Moreover, CiPSCs weremaintained with normal karyotypes (data not shown). Together, theseresults establish a robust CiPSC induction protocol, obtained bymanipulating the cell fate transition more precisely through theXEN-like state.

Gene Expression Dynamics During CiPSC Generation

Expression of some typical pluripotency-associated genes during chemicalreprogramming was examined at different stages of chemicalreprogramming. The data showed sequential expression of pluripotencygenes (FIG. 20A). Single cell qRT-PCR analysis showed that approximately50% of the cells in stage 2 co-expressed XEN cell markers (FIG. 20B).Similar to embryo-derived XEN cells, the XEN-like cells formed duringchemical reprogramming expressed several pluripotency genes, such asSall4 and Lin28a, during the early stages of reprogramming (Lim et al.,Cell Stem Cell, 3:543-554 (2008); McDonald et al., Cell Reports,9:780-793 (2014). During an extended time in culture, the XEN-like cellsexpressed other pluripotency-associated genes on days 16-28, such asOct4 and Dppa2. During stage 3, the expression of most pluripotencymarker genes, including Nanog, was activated in CiPSCs (FIGS. 20A and20C). This finding indicates a process of sequential gene activationfrom XEN-like cells to pluripotent stem cells. Interestingly, Sall4,Lin28a, Esrrb, the major genes associated with pluripotency that arehighly expressed during stages 1 and 2 of cell reprogramming, havepreviously been reported to be predictive markers of transcriptionfactor-induced reprogramming and to be sufficient for inducing iPSCswith high quality when concomitantly expressed with Nanog (Buganim etal., Cell, 150:1209-1222 (2012); Buganim et al., Cell Stem Cell,15:295-309 (2014)).

Through qRT-PCR analysis and RNA sequencing, AM580 and EPZ004777 wereidentified as molecules which both promote the expression of XEN markergenes, such as Sall4, Gata4 and Sox17, during stage 1 of chemicalreprogramming from fibroblasts to XEN-like cells (FIG. 17F). SGC0946 and5-aza-dC promote the expression of pluripotency genes, such as Oct4 andDppa family genes in XEN-like cells, during stage 2 of chemicalreprogramming (FIG. 20D). These findings indicate that stage 1 ofchemical reprogramming into XEN-like cells is promoted by additionalsmall molecules that act by enhancing the expression of XEN mastergenes, and stage 2 can be shortened possibly due to the enhancedactivation of pluripotency-associated genes by additional smallmolecules. Studies were next conducted to determine whether thereprogramming process through a XEN-like state is a unique route towardspluripotency compared to that of the transgenic strategy, which usesOSKM (Takahashi et al., Cell, 131:861-872 (2007); Takahashi et al.,Nature Communications 5:3678 (2014); Takahashi and Yamanaka, Cell,126:663-676 (2006). Notably, OSKM-induced reprogramming processes do notshow XEN-like gene profiles, analyzed by qRT-PCR (FIG. 20C). Thisfinding was also consistent with the original data from RNA sequencingor microarray during the reprogramming process in other reports(Golipour et al., Cell Stem Cell 11:769-782 (2012); Mikkelsen et al.,Nature, 454:49-55 (2008); Polo et al., Cell 151:1617-1632 (2012);Sridharan et al., Cell 136:364-377 (2009).

Also examined was whether primitive streak genes were expressed duringthe chemical reprogramming process, because a primitive streak state hasbeen reported during the process of OSKM-induced reprogramming(Takahashi et al., Nature Communications, 5:3678 (2014)). The expressionof primitive streak markers, such as T and Mixl1, was not detectedduring chemical reprogramming (data not shown), demonstrating a uniqueXEN-like state during the chemical reprogramming process, which differsfrom that of the reprogramming induced by transgenes (FIG. 20E).

XEN Master Gene Expression is Essential During Chemical Reprogramming

To further support the XEN state as an intermediate for chemicalreprogramming and to understand the role of XEN master genes in chemicalreprogramming, knockdown and ectopic expression experiments wereperformed. Knockdown of any one of the key XEN genes Sall4, Gata4, Gata6or Sox/7 led to a significant down regulation in the mRNA levels of theother XEN genes and decreased XEN-like colony numbers, thus resulting inless Oct4 expression and fewer CiPSCs at the end of the reprogrammingperiod (FIGS. 21A-E). The expression of XEN genes was essential to Oct4expression. XEN-gene expression was also enhanced in chemicalreprogramming from neural stem cells and intestinal epithelium cells(data not shown), and the knockdown of these genes impaired XEN-likecolony formation and further CiPSC induction from these two initial celltypes (FIG. 21F). These results further indicate that the XEN-like stateis essential to the chemical reprogramming process. In contrast, theseXEN master genes, such as Gata4, Gata6 and Sox17, were not required inOSKM-induced reprogramming (FIG. 21G), which suggest different roadmapsunderlying chemical reprogramming and OSKM-induced reprogramming (20E).

Furthermore, the overexpression of two of the XEN master genes (SALL4plus GATA4 or SALL4 plus GATA6) in fibroblasts sufficed in inducingXEN-like colony formation in the absence of the three key smallmolecules, CHIR99021, 616452 and Forskolin (FIG. 21H). The resultingXEN-like cells showed gene expression pattern similar to that of thesmall molecule-induced XEN-like cells (FIG. 21I). Moreover, Oct4expression was detected in the XEN-like colonies induced by the twocombinations of XEN cell master transcription factors (FIG. 21J and datanot shown). These findings suggest that XEN genes are both necessary andsufficient to initiate the expression of Oct4, a master gene ofpluripotency.

Notably, although these XEN master gene-induced XEN-like cells expressedOct4, they could not be further reprogrammed into iPSCs, even with aprolonged culture in 2i-medium. Exogenous XEN genes downregulated theendogenous expression of Sox2 (FIG. 21K).

Accordingly, when Sox2 was exogenously provided in an appropriate timewindow after XEN gene overexpression, iPSCs were obtained (FIG. 21L anddata not shown). This finding is consistent with our previous findingsof a seesaw model in regulating pluripotency establishment, in which theGata family genes and Sox2 should be in a balance to achievepluripotency (Shu et al., Cell, 153:963-975 (2013), whereas in chemicalreprogramming such a balance could be a dynamic process rather than asteady equilibrium.

The XEN-Like Intermediates Resemble Embryo-Derived XEN Cells in GeneExpression Patterns, In Vivo Development Potential and ReprogrammingPotential

Chemically-induced XEN-like cells were compared to embryo-derived XENcells (eXENs) (Kunath et al., Development 132:1649-1661 2005) withrespect to in vitro culture conditions. The studies showed that XEN-likecells could not be maintained in the traditional XEN culture medium(RPMI CM (traditional XEN culture medium; RPMI-medium with 70%MEF-condition medium (Kunath, et al., Development, 132:1649-1661 (2005))(data not shown). In addition, eXEN cell lines could be maintainedlong-term and expanded in the stage 1 medium of chemical reprogrammingfor more than 20 passages, with the gene expression pattern and in vivodevelopment potential similar to eXENs maintained in traditional XENmedium (data not shown).

By using stage 1 medium including chemical cocktail VC6TF,chemically-derived eXEN cell lines (CeXENs) could be establisheddirectly from blastocysts and expanded long-term for more than 25passages, with a XEN-like gene expression pattern and in vivointegration capability into extraembryonic parietal endoderm (FIGS.22A-B, Table 7, and data not shown).

TABLE 7 Statistical table of XEN integration chimeric ability ofdifferent cell types as indicated to parietal endoderm. EGFP-labeledfibroblasts were set as negative control. No. of embyos recovered CellType (E6.5-8.5) Xen integration Xen-like D11 18  7 (39%) Xen-like D11624 21 (88%) Xen-like D125 22 10 (45%) eXen-1 31 20 (65%) eXen-2 23  9(39%) CeXen 20  7 (35%) Fibroblasts 10  0

qRT-PCR analysis showed that XEN-like cells express a comparable levelof XEN master genes to that of eXENs and CeXEN (FIGS. 16B, 22A and datanot shown). Global gene expression profiling at the end of stages 1 and2 showed that XEN-like cells showed gene expression profiles close toeXENs and CeXENs (FIG. 22B). Principal component analysis (PCA) analysisof gene expression profiling showed a clear roadmap from fibroblaststoward pluripotent stem cells through such a XEN-like state close toeXENs and CeXENs (data not shown).

In particular, the XEN-like intermediates in chemical reprogramming werecloser to CeXENs than traditional eXENs in gene expression profiles(FIG. 22B and data not shown). Moreover, mRNA level of EpCAM, Cdh1 andSox2 in XEN-like cells and CeXENs was notably higher than that intraditional eXENs (FIG. 22C). It is possible that the differences of thegene expression pattern between XEN-like cells and traditional eXENswere resulted from their different culture conditions. Interestingly,authentic XEN cells in vivo express EPCAM and CDH1 in a high levelcomparable to that of ESCs and express SOX2 in a relatively low level, apattern similar to that of XEN-like cells and CeXENs, but not eXENs(FIG. 22D). This suggests that although XEN-like cells showed somedifferences to eXENs in culture conditions and gene expression patterns,they were more similar to CeXEN, another type of embryo-derived XENcells.

The in vivo development potential of XEN-like cells during chemicalreprogramming was also examined. XEN-like cells induced in differenttime courses of chemical reprogramming were injected into mouseblastocysts. Similarly to eXEN cells, XEN-like cells at days 11-25 ofchemical reprogramming were able to integrate into the parietal endodermof the extraembryonic tissues with a comparable efficiency of eXENs,without any integration in the embryos (data not shown and Table 7). Inparticular, XEN-like cells in day 16 of chemical reprogramming showedthe highest ratio of XEN integration (Table 7). These findings suggestthat XEN-like cells resemble embryo-derived XEN cells in terms ofdevelopment potential. Furthermore, either eXENs derived by traditionalXEN culture medium or CeXENs established by the stage 1 medium ofchemical reprogramming, were capable of further reprogramming intoCiPSCs by using the protocol of late chemical reprogramming in stages 2and 3 (data not shown). Notably, 17-34 CiPSC colonies were generatedfrom 2,000 CeXENs within 24 days, a reprogramming efficiency even higherthan that of XEN-like cells.

CiPSCs generated from eXENs and CeXENs were further characterized topossess an expression pattern similar to pluripotency stem cells (FIG.22D and data not shown). These findings further support that XEN-likecells induced in the early stage of CiPSC generation, which were similarto CeXENs, were amenable to being further reprogrammed in the late stageof chemical reprogramming.

DISCUSSION

In summary, we demonstrated a XEN-like state as an intermediate forchemical reprogramming, which differs from other reprogrammingscenarios. The chemical reprogramming process can be divided into twomajor steps. In the first step of reprogramming, fibroblasts aredirectly converted into XEN-like cells, whereas in the later step ofreprogramming, XEN-like cells are converted into CiPSCs (FIG. 15D).

The determination of the role of a XEN-like state in chemicalreprogramming uncovered a unique route in chemical reprogramming ofsomatic cells toward pluripotency, but not in OSKM-induced reprogramming(FIG. 20E). This route also differs from a primitive streak-like statemediated reprogramming process induced by transcription factors(Takahashi et al., Nature Communications 5:3678 (2014)). In addition,fibroblasts from mouse embryos were induced into a cell type resemblingextraembryonic lineages in chemical reprogramming process, which isdefinitely not a reversed normal development process with multipletransient waves of gene expression changes as recently reported(Cacchiarelli et al., Cell 162:412-424 (2015)). The XEN-like state isalso required to generate CiPSCs from other cell types, such as neuralstem cells and intestinal epithelium cells, indicative of a generalpathway to reprogram other cell types using small molecules.Interestingly, the XEN-like cells induced during chemical reprogrammingare similar to embryo-derived XEN cells in gene expression pattern,development potential and reprogramming potential. These in togetherprovide a new framework to further study cell fate determination of XENcells and pluripotent cells and the transition of these cell fates.

Moreover, the XEN-like state is unique, as it is primed to undergofurther conversion to the pluripotency state. Similarly to XEN cells invivo, the induced XEN-like cells have already expressed Sall4 andLin28a, two master genes of pluripotency. It is possible that the sharedgenes that are expressed in both XEN cells and pluripotent stem cells,such as Sall4 and Lin28a, make the pluripotency state more accessibleduring the cell fate transition from the XEN-like state to pluripotency.Moreover, sequential expression of many pluripotency marker genes inXEN-like cells, during stage 2 of chemical reprogramming was alsodemonstrated. For example, the expression of Esrrb and Oct4 wasactivated, and the expression of Oct4 was compatible with the expressionof XEN genes. This finding indicates that the pluripotency network iseasily established in XEN-like cells. This finding is consistent withthe recent report that in vivo XEN cells spontaneously transition intoepiblast stem cells, which are in a primed pluripotency state(Xenopoulos et al., Cell Reports, 10:1508-1520 (2015). The compatibilityof XEN-like genes and the expression of pluripotency genes make theXEN-like state an ideal bridge between somatic cells and pluripotentcells.

These studies establish that XEN-like state is essential for inducingpluripotency during chemical reprogramming, possibly because that themaster genes of XEN directly contribute to the establishment ofpluripotency. In addition, the studies suggest that XEN-like genes mayhave dual roles in chemical reprogramming. As previously reported, thereare several mutual antagonistic mechanisms between XEN genes andpluripotency-associated genes, such as the incompatibility between Sox17and Sox2 and between Gata6 and Nanog (Aksoy et al., The EMBO Journal32:938-953 (2013); Chazaud et al., Developmental Cell, 10:615-624 (2006;Niakan et al., Genes & Development, 24:312-326 (2010). In this study,the expression of Sox2 was repressed by the XEN genes. Further, duringstage 3 of chemical reprogramming, the expression of Nanog and Sox2 wasincompatible with the expression of Gata4, reminiscent of the cell fatedetermination between XENs and epiblasts regulated by FGF/ERK signalingin the mouse blastocyst as previously reported (Yamanaka et al.,Development, 137:715-724 (2010). During the early stage of chemicalreprogramming, the XEN state is necessary to initiate the expression ofselect pluripotency genes, such as Sall4, Lin28a and Oct4. However, inthe later stage of reprogramming, these XEN genes need to be silenced toinitiate the expression of additional pluripotency genes, such as Nanogand Sox2.

Most important, in this study, chemical reprogramming was greatlyimproved by manipulating cell fate transitions through the XEN-likestate, through careful selection the small-molecule cocktails which biascells towards the XEN-like state, and then CiPSC, concentrations,durations and other details of cell manipulation during each stage,greatly improving the efficiency of CiPSC generation and increasing thetotal yields of CiPSC colonies by up to 1,000-fold compared a chemicalreprogramming protocol which does not proceed via biasing to a XEN-likestate as described herein.

1. A kit or cell culture media composition for inducing pluripotency innon-pluripotent eukaryotic cells, the composition comprising chemicalinducers of pluripotency (CIPs) from each of the following groups (1)glycogen synthase kinase (GSK) inhibitors, (2) TGFβ receptor inhibitors,(3) cyclic AMP (cAMP) agonists, (4) S-adenosylhomocysteine hydrolase(SAH) inhibitors, (5) histone acetylators, (6) DOT1L methyltransferaseinhibitors, (7) Retinoic acid receptor (RAR) agonist, (8) epigeneticmodulator, and (9) inhibitors of histone demethylation in amountseffective to induce reprogramming of XEN-like cells into pluripotentcells.
 2. The kit or composition of claim 1, wherein the GSK inhibitoris[6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile](“C”); the TGFβ receptor inhibitor is[2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine] (“6”);the cAMP agonist is Forskolin (“F”), the SAH inhibitor is3-deazaneplanocin A (“Z”), the DOT1L methyltransferase inhibitor is SGC0946 (“S”) (1-[3-[[[(2R,3S,4R,5R)-5-(4-Amino-5-bromo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methyl](isopropyl)amino]propyl]-3-[4-(2,2-dimethylethyl)phenyl]urea)or EPZ004777,“1-(3-((((2R,3S,4R,5R)-5-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)(isopropyl)amino)propyl)-3-(4-(tert-butyl)phenyl)urea(“E”); RAR agonists include AM 580 (“A”)(4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoicacid); the epigenetic modulator is 5-azacytidine (“D”) and the inhibitorof histone demethylation is tranylcypromine (“T”) and optionally,wherein the composition comprises 2i-medium, the 2i medium optionallyfurther comprising N2B27.
 3. The kit or composition of claim 2comprising VC6TFZASD.
 4. (canceled)
 5. The kit or composition of claim1, further comprising a least one small molecule selected from the groupconsisting of: (i) small molecules that facilitate late reprogramming;(ii) epigenetic modulators selected from the group consisting of sodiumbutyrate and RG108 [N-Phthalyl-L-tryptophan] (iii) small molecules thatimprove/boost chemical reprogramming efficiency selected from the groupconsisting of SF1670[N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide]; DY131[N-(4-(Diethylaminobenzylidenyl)-N-(4-hydroxybenzoyl)-hydrazine];UNC0638[2-Cyclohexyl-6-methoxy-N-[1-(1-methylethyl)-4-piperidinyl]-7-[3-(1-pyrrolidinyl)propoxy]-4-quinazolinamine];SRT1720[N-(2-(3-(piperazin-1-ylmethyl)imidazo[2,1-b]thiazol-6-yl)phenyl)quinoxaline-2-carboxamidehydrochloride]; 2-Me-5HT (2-methyl-5-hydroxytryptamine); IBMX[3,7-Dihydro-1-methyl-3-(2-methylpropyl)-1H-purine-2,6-dioneand];(4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide)and TTNPB[4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid and (iv) protein growth factors.
 6. The composition of claim 5wherein the late reprogramming facilitator is selected from the groupconsisting of prostaglandin E2 (PGE2) and rolipram. 7-10. (canceled) 11.The kit of claim 2 in a kit, wherein the small molecular weightcompounds are present in relative amounts to put into cell culture mediafor differentiated cells to induce expression of XEN markers selectedfrom the group consisting of SALL4, GATA4 and SOX1.
 12. A method ofinducing pluripotency in partially or completely differentiated cells,the method comprising: (a) contacting the cell to be reprogrammed with afirst cocktail of CIPs (XEN-Cocktail) for a sufficient period of time tobias the cells into a XEN-like cell population; (b) contacting thepopulation of XEN-like cells for a sufficient period of time toreprogram the cell into a chemically induced pluripotent stem cell(CiPSC) with a second cocktail of CIPS (herein, XEN-CiPSC cocktail); and(c) culturing the cells in 2i-medium.
 13. The method of claim 12,wherein the differentiated cells are selected from the group consistingof multipotent stem cells, cells of hematological origin, cells ofembryonic origin, skin derived cells, fibroblasts, adipose cells,epithelial cells, endothelial cells, mesenchymal cells, parenchymalcells, neurological cells, and connective tissue cells.
 14. The methodof claim 14, wherein the cells to be induced are selected from the groupconsisting of fibroblasts, adipose-derived cells, neural derived cellsand intestinal epithelial cells, preferably not expressing Oct4 andoptionally, wherein the cells are not transfected to express any ofOct4, KLF4, SOX2, C-Myc or NANOG.
 15. (canceled)
 16. The method of claim12, wherein the cells to be induced are cultured in reprogramming mediumcomprising the CIPs for a period between 26-30 days.
 17. The method ofclaim 12 wherein the cell culture medium comprises VC6TFA the entiretime before the use of 2i-medium.
 18. The method of claim 12 wherein thecells are replated at a density between 50,000 to 100,000 per well in a6-well plate between day 6-10.
 19. The method of claim 12 wherein theXEN-cocktail comprises VC6TFAE and/or the XEN-CiPSC cocktail comprisesVC6TFZASD.
 20. (canceled)
 21. The method of claim 12 wherein the mediumis changed into 2i-medium between day 26 to and day 30 and cultured in2i-medium for about 10-14 days and optionally, wherein the 2i-mediumfurther comprises N2B27. 22-23. (canceled)
 24. The method of claim 12wherein the cell culture medium further comprising small molecules thatfacilitate late reprogramming selected from the group consisting of cAMPagonists, epigenetic modulators and small molecules that improve/boostchemical reprogramming efficiency.
 25. The method of claim 12 whereinthe XEN-cocktail does not comprise SGC
 0946. 26. The method of claim 12,wherein the media comprising the small molecule that boostsreprogramming efficiency, TTNPB.
 27. The method of claim 12, comprisingidentifying the pluripotent cells based on morphology, doubling time,the ability of the cell to differentiate into tissues of the threeembryonic germ layers, expression of ESC markers and combinationsthereof wherein the ESC markers are selected from the group consistingof alkaline phosphatase (AP); nanog; Rex1; Sox2; Dax1; Sall4;undifferentiated embryonic cell transcription factor (Utf1); stagespecific embryonic antigen-4 (SSEA-4) and combinations thereof; andoptionally, isolating the pluripotent cells. 28-29. (canceled) 30.Pluripotent cells obtained by the method of claim
 12. 31. A therapeuticcomposition comprising the pluripotent cells of claim 30, formulated foradministration to an individual by injection, implantation of aprosthetic device or tissue engineering matrix.
 32. The pluripotentcells of claim 31, wherein the the ESC markers are selected from thegroup consisting of alkaline phosphatase (AP); nanog; Rex1; Sox2; Dax1;Sall4; undifferentiated embryonic cell transcription factor (Utf1);stage specific embryonic antigen-4 (SSEA-4) and combinations thereof;and optionally, isolating the pluripotent cells.